Novel polyketide derivatives

ABSTRACT

The present invention relates to novel polyketides, host cells that produce the novel compounds, and methods fort their use. The compounds of the present invention are cyclic polyketides (also referred to as a “macrolides” or “macrolactones”) that include  
                 
 
     as part of their structure and bind to a FK binding protein wherein R 4  and R 5  are each selected from the group consisting of hydrogen, methyl, ethyl, and methoxy, provided that at least one of R 4  and R 5  is hydrogen, methyl, or ethyl. As will be explained in greater detail below, the compounds of the present invention have properties such as favorable P450 enzyme activity profiles that are desirable for use of these compounds as drugs.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application asserts priority to (i) U.S. patentapplication Ser. No. 09/410,551 filed Oct. 1, 1999 by inventorsChristopher Reeves, Daniel Chu, Chaitan Khosla, Daniel Santi, and Kai Wuentitled POLYKETIDE SYNTHASE ENZYMES AND RECOMBINANT DNA CONSTRUCTSTHEREFOR and to U.S. provisional applications (ii) Serial No. 60/252,968filed Nov. 22, 2000 by inventors Daniel Chu, Chaitan Khosla, DanielSanti and Maria Fardis entitled NOVEL POLYKETIDE DERIVATIVES (iii)Serial No. 60/218,176 filed Jul. 14, 2000 by inventors Daniel Chu,Chaitan Khosla, Daniel Santi, and Maria Fardis entitled METABOLICALLYSTABLE DERIVATIVES OF FK-506 AND FK-520 and (iv) Serial No. 60/204,828filed May 17, 2000 by inventors Daniel Chu, Christopher Reeves, ChaitanKhosla, Daniel Santi and Maria Fardis entitled NOVEL POLYKETIDEDERIVATIVES, all of which are incorporated herein by reference in theirentireties.

GOVERNMENT SUPPORT

[0002] This invention was made in whole or in part with governmentsupport from National Institute of Allergy and Infectious Diseases underSBIR Grant No. 1R43 AI46206-01-A1. Accordingly, the government may havecertain rights in the invention.

BACKGROUND

[0003] Polyketides are a diverse class of compounds that are the sourceof many biologically active molecules such as tetracycline,erythromycin, epothilone, narbomycin, picromycin, rapamycin, spinocyn,and tylosin. Other important examples include naturally occurringimmunosuppressants FK-506 (also known as tacrolimus) and FK-520 (alsoknown as ascomycin).

[0004] Differing by a single substituent (R¹) at C-21, FK-506 has anallyl group whereas FK-520 has an ethyl group at this position. Of thetwo, FK-506 has been particularly well studied and is used currently asan immunosuppressive drug.

[0005] FK-506 and FK-520 exert their biologic effects through theinitial formation of an intermediate complex with proteins known asFKBPs (FK-506 binding proteins) such as FKBP-12 and FKBP-52. Theseproteins are a class of cytosolic proteins that form complexes withmolecules such as FK-506, FK-520, and rapamycin that in turn serve asligands for other cellular targets involved in signal transduction.Binding of FK-506, FK-520, and rapamycin to FKBP occurs through thestructurally similar segments of the polyketide molecules, known as the“FKBP-binding domain” (as generally but not precisely indicated by thestippled regions in the structures below).

[0006] The FK-506-FKBP complex then binds calcineurin, while therapamycin-FKBP complex binds to a protein known as RAFT-1. Binding ofthe FKBP-polyketide complex to these second proteins occurs through thedissimilar regions of the drugs known as the “effector” domains.

[0007] The three component FKBP-polyketide-effector complex is requiredfor signal transduction and subsequent immunosuppressive activity ofFK-506, FK-520, and rapamycin. Modifications in the effector domains ofFK-506, FK-520, and rapamycin that destroy binding to the effectorproteins (calcineurin or RAFT) but leave FKBP binding unaffected lead toloss of immunosuppressive activity. Further, such analogs antagonize theimmunosuppressive effects of the parent polyketides, because theycompete for FKBP. Such non-immunosuppressive analogs also show reducedtoxicity (see Dumont et al., 1992, Journal of Experimental Medicine 176,751-760), indicating that much of the toxicity of these drugs is througha mechanism not mediated by FKBP binding.

[0008] In addition to immunosuppressive activity, FK-520, FK-506, andrapamycin have neurotrophic activity. In the central nervous system andin peripheral nerves, the corresponding target proteins are referred toas neuroproteins. The neuro-FKBP is markedly enriched in the centralnervous system and in peripheral nerves. Molecules that bind to theneuro-FKBP, such as FK-506 and FK-520, have the remarkable effect ofstimulating nerve growth. In vitro, they act as neurotrophins. Moreparticularly, they promote neurite outgrowth in NGF-treated PC12 cellsand in sensory neuronal cultures, and they promote regrowth of damagedfacial and sciatic nerves, and repair lesioned serotonin and dopamineneurons in the brain in intact animals. See Gold et al., June 1999, J.Pharm. Exp. Ther. 289(3): 1202-1210; Lyons et al., 1994, Proc. NationalAcademy of Science 91: 3191-3195; Gold et al., 1995, Journal ofNeuroscience 15: 7509-7516; Steiner et al., 1997, Proc. National Academyof Science 94: 2019-2024; and U.S. Pat. Nos. 5,968,921 and 6,210,974.Further, the restored central and peripheral neurons appear to befunctional.

[0009] Compared to protein neurotrophic molecules (e.g., BNDF, NGF,etc.), the small-molecule neurotrophins such as FK-506, FK-520, andrapamycin have different, and often advantageous, properties. First,whereas protein neurotrophins are difficult to deliver to their intendedsite of action and may require intra-cranial injection, thesmall-molecule neurotrophins display excellent bioavailability; they areactive when administered subcutaneously and orally. Second, whereasprotein neurotrophins show quite specific effects, the small-moleculeneurotrophins show rather broad effects. Finally, whereas proteinneurotrophins often show effects on normal sensory nerves, thesmall-molecule neurotrophins do not induce aberrant sprouting of normalneuronal -processes and seem to affect damaged nerves specifically.Neuro-FKBP ligands have therapeutic utility in a variety of disordersinvolving nerve degeneration (e.g. multiple sclerosis, Parkinson'sdisease, Alzheimer's disease, stroke, traumatic spinal cord and braininjury, peripheral neuropathies).

[0010] The metabolism and pharmacokinetics of FK-506 have beenextensively studied, and FK-520 is believed to be similar in theserespects. Absorption of FK-506 is rapid, variable, and incomplete fromthe gastrointestinal tract (Harrison's Principles of Internal Medicine,14th edition, 1998, McGraw Hill, 14, 20, 21, 64-67). The meanbioavailability of the oral dosage form is 27% (range 5 to 65%). Thevolume of distribution (VolD) based on plasma is 5 to 65 L per kg ofbody weight (L/kg), and is much higher than the VolD based on wholeblood concentrations, the difference reflecting the binding of FK-506 tored blood cells. Whole blood concentrations may be 12 to 67 times theplasma concentrations. Protein binding is high (75 to 99%), primarily toalbumin and alpha1-acid glycoprotein. The half-life for distribution is0.9 hour; elimination is biphasic and variable: terminal—11.3 hours(range, 3.5 to 40.5 hours). The time to peak concentration is 0.5 to 4hours after oral administration.

[0011] FK-506 is metabolized primarily by cytochrome P450 3A enzymes inthe liver and small intestine. The drug is extensively metabolized withless than 1% excreted unchanged in urine. Because hepatic dysfunctiondecreases clearance of FK-506, doses have to be reduced substantially inprimary graft non-function, especially in children. In addition, thebioactivity of FK-506 is affected by drugs that modulate the activity ofP450 3A enzymes. Drugs that induce the cytochrome P450 3A enzymes reduceFK-506 levels, while drugs that inhibit these P450s increase FK-506levels. For example, FK-506 bioavailability doubles withco-administration of ketoconazole, a drug that inhibits P450 3A. See,Vincent et al., 1992, Arch. Biochem. Biophys. 294: 454-460; Iwasaki etal., 1993, Drug Metabolism & Disposition 21: 971-977; Shiraga et al.,1994, Biochem. Pharmacol. 47: 727-735, and Iwasaki et al., 1995, DrugMetabolism & Disposition 23: 28-34.

[0012]FIG. 1 shows the eight isolated metabolic products formed fromincubation of FK-506 with liver microscomes. As can be seen, fourmetabolites of FK-506 involve demethylation of the methoxy groups oncarbons 13, 15, and 31, and hydroxylation of carbon 12. The13-demethylated (hydroxy) compounds undergo cyclizations of the13-hydroxy at carbon 10 to give M-I, M-VI and M-VII, and the 12-hydroxymetabolite at carbon 10 to give M-I. Another four metabolites formed byoxidation of the four metabolites mentioned above were isolated by livermicrosomes from dexamethasone treated rats. Three of these aremetabolites doubly demethylated at the methoxy groups on carbons 15 and31 (M-V), 13 and 31 (M-VI), and 13 and 15 (M-VII). The fourth, M-VIII,was the metabolite produced after demethylation of the carbon 31-methoxygroup, followed by formation of a fused ring system by furtheroxidation. Among the eight metabolites, M-II has immunosuppressiveactivity comparable to that of FK-506, whereas the other metabolitesexhibit weak or negligible activities. Importantly, the major metaboliteof human, dog, and rat liver microsomes (representing approximatelyabout 90% of the metabolic products after a 10 minute incubation) is the13-demethylated and cyclized FK-506 (M-I).

[0013] A disadvantage of using FK-506 and FK-520 as drugs is dosingunpredictability. Due to the significant variability in metabolism amongpatients, an appropriate dosing regimen is difficult-to ascertain for anindividual patient. Another disadvantage of FK-506 and FK-520 is theirdual pharmacological effects as immunosuppressants and as neurotrophicagents. In general, compounds having a single specificity are desired.For example, a FK-506 like compound having only neurotrophic activitywithout immunosuppressive activity or vice versa may be used to treatthe intended symptom without the side effects of the other bioactivity.

[0014] As a result, derivatives that improve upon the properties ofFK-506 and FK-520 are needed and desired. However, because FK-506 andFK-520 are complex structures that are generally not amenable to eitherde novo chemical synthesis or facile derivation, this need remainsunfulfilled.

SUMMARY

[0015] The present invention relates to novel polyketides, host cellsthat produce the novel compounds, and methods fort their use. Thecompounds of the present invention are cyclic polyketides (also referredto as a “macrolides” or “macrolactones”) that include

[0016] as part of their structure and bind to a FK binding proteinwherein R⁴ and R⁵ are each selected from the group consisting ofhydrogen, methyl, ethyl, and methoxy, provided that at least one of R⁴and R⁵ is hydrogen, methyl, or ethyl. As will be explained in greaterdetail below, the compounds of the present invention have propertiessuch as favorable P450 enzyme activity profiles that are desirable foruse of these compounds as drugs.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1 shows the proposed degradative pathway for FK-506metabolism.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The present invention relates to novel polyketides structurallyrelated to FK-506 and FK-520 and to methods for making and using thesame.

[0019] Definitions

[0020] The following general statements and terms are used to describethe compounds of the present invention.

[0021] All stereoisomers of the inventive compounds are included withinthe scope of the invention, as pure compounds as well as mixturesthereof. Individual enantiomers, diastereomers, geometric isomers, andcombinations and mixtures thereof are all encompassed by the presentinvention. Furthermore, some of the crystalline forms for the compoundsmay exist as polymorphs and as such are included in the presentinvention. In addition, some of the compounds may form solvates withwater (i.e., hydrates) or common organic solvents, and such solvates arealso encompassed within the scope of this invention.

[0022] Protected forms of the inventive compounds are included withinthe scope of the present invention. A variety of protecting groups aredisclosed, for example, in T. H. Greene and P. G. M. Wuts, ProtectiveGroups in Organic Synthesis, Third Edition, John Wiley & Sons, New York(1999), which is incorporated herein by reference in its entirety. Forexample, a hydroxy protected form of the inventive compounds are thosewhere at least one of the hydroxyl groups is protected by a hydroxyprotecting group. Illustrative hydroxyl protecting groups include butnot limited to tetrahydropyranyl; benzyl; methylthiomethyl;ethylthiomethyl; pivaloyl; phenylsulfonyl; triphenylmethyl;trisubstituted silyl such as trimethyl silyl, triethylsilyl,tributylsilyl, tri-isopropylsilyl, t-butyldimethylsilyl,tri-t-butylsilyl, methyldiphenylsilyl, ethyldiphenylsilyl,t-butyldiphenylsilyl, and the like; acyl and aroyl such as acetyl,pivaloylbenzoyl, 4-methoxybenzoyl, 4-nitrobenzoyl and aliphatic acylaryland the like. Keto groups in the inventive compounds may similarly beprotected.

[0023] The present invention includes within its scope prodrugs of thecompounds of this invention. In general, such prodrugs are functionalderivatives of the compounds that are readily convertible in vivo intothe required compound. Thus, in the methods of treatment of the presentinvention, the term “administering” shall encompass the treatment of thevarious disorders described with the compound specifically disclosed orwith a compound which may not be specifically disclosed, but whichconverts to the specified compound in vivo after administration to asubject in need thereof. Conventional procedures for the selection andpreparation of suitable prodrug derivatives are described, for example,in “Design of Prodrugs”, H. Bundgaard ed., Elsevier, 1985.

[0024] As used herein, the term “aliphatic” refers to saturated andunsaturated straight chained, branched chain, cyclic, or polycyclichydrocarbons that may be optionally substituted at one or morepositions. Illustrative examples of aliphatic groups include alkyl,alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties.The term “alkyl” refers to straight or branched chain saturatedhydrocarbon substituent. “Alkenyl” refers to a straight or branchedchain hydrocarbon substituent with at least one carbon-carbon doublebond. “Alkynyl” refers to a straight or branched chain hydrocarbonsubstituent with at least one carbon-carbon triple bound.

[0025] The term “aryl” refers to monocyclic or polycyclic groups havingat least one aromatic ring structure that optionally include one oremore heteroatoms and preferably include three to fourteen carbon atoms.Aryl substituents may optionally be substituted at one or morepositions. Illustrative examples of aryl groups include but are notlimited to: furanyl, imidazolyl, indanyl, indenyl, indolyl, isooxazolyl,isoquinolinyl, naphthyl, oxazolyl, oxadiazolyl, phenyl, pyrazinyl,pyridyl, pyrimidinyl, pyrrolyl, pyrazolyl, quinolyl, quinoxalyl,tetrahydronaphththyl, tetrazolyl, thiazolyl, thienyl, and the like.

[0026] The term “heteroaryl” is an aryl that includes one or more heteroatoms such as O, N, and S.

[0027] The aliphatic (i.e., alkyl, alkenyl, etc.) and aryl moieties maybe optionally substituted with one or more substituents, preferably fromone to five substituents, more preferably from one to threesubstituents, and most preferably from one to two substituents. Thedefinition of any substituent or variable at a particular location in amolecule is independent of its definitions elsewhere in that molecule.It is understood that substituents and substitution patterns on thecompounds of this invention can be selected by one of ordinary skill inthe art to provide compounds that are chemically stable and that can bereadily synthesized by techniques known in the art as well as thosemethods set forth herein. Examples of suitable substituents include butare not limited to: alkyl, alkenyl, alkynyl, aryl, halo;trifluoromethyl; trifluoromethoxy; hydroxy; alkoxy; cycloalkoxy;heterocyclooxy; oxo; alkanoyl (—C(═O)-alkyl which is also referred to as“acyl”)); aryloxy; alkanoyloxy; amino; alkylamino; arylamino;aralkylamino; cycloalkylamino; heterocycloamino; disubstituted amines inwhich the two amino substituents are selected from alkyl, aryl, oraralkyl; alkanoylamino; aroylamino; aralkanoylamino; substitutedalkanoylamino; substituted arylamino; substituted aralkanoylamino;thiol; alkylthio; arylthio; aralkylthio; cycloalkylthio;heterocyclothio; alkylthiono; arylthiono; aralkylthiono; alkylsulfonyl;arylsulfonyl; aralkylsulfonyl; sulfonamido (e.g., SO₂NH₂); substitutedsulfonamido; nitro; cyano; carboxy; carbamyl (e.g., CONH₂); substitutedcarbamyl (e.g., —C(═O)NRR′ where R and R′ are each independentlyhydrogen, alkyl, aryl, aralkyl and the like); alkoxycarbonyl, aryl,substituted aryl, guanidino, and heterocyclo such as indoyl, imidazolyl,furyl, thienyl, thiazolyl, pyrrolidyl, pyridyl, pyrimidyl and the like.Where applicable, the substituent may be further substituted such aswith, alkyl, alkoxy, aryl, aralkyl, halogen, hydroxy and the like.

[0028] The terms “alkylaryl” or “arylalkyl” refer to an aryl group withan aliphatic substituent that is bonded to the compound through thealiphatic group. An illustrative example of an alkylaryl or arylalkylgroup is benzyl, a phenyl with a methyl group that is bonded to thecompound through the methyl group (—CH₂Ph where Ph is phenyl).

[0029] The term “alkoxy” refers to —OR wherein O is oxygen and R is analiphatic group.

[0030] The term “hydroxyalkyl” refers to —ROH where R is an aliphaticmoiety.

[0031] In addition to the explicit substitutions at the above-describedgroups, the inventive compounds may include other substitutions whereapplicable. For example, the lactone backbone or backbone substituentsmay be additionally substituted (e.g., by replacing one of the hydrogensor by derivatizing a non-hydrogen-group) with one or more substituentssuch as C₁-C₅ aliphatic, C₁-C₅ alkoxy, aryl, or a functional group.Illustrative examples of suitable functional groups include but are notlimited to: acetal, alcohol, aldehyde, amide, amine, boronate,carbamate, carboalkoxy, carbonate, carbodiimide, carboxylic acid,cyanohydrin, disulfide, enamine, ester, ether, halogen, hydrazide,hydrazone, imide, imido, imine, isocyanate, ketal, ketone, nitro, oxime,phosphine, phosphonate, phosphonic acid, quaternary ammonium, sulfenyl,sulfide, sulfone, sulfonic acid, thiol, and the like.

[0032] The term “FKBP” refers to a protein (of greater than 90% purity)that binds FK-506 with a K_(d) (equilibrium binding constant) that isapproximately equal to or less than about 1 μM in an in vitro assay.Illustrative examples of FKBPs include but are not limited to FKBP-12,(U.S. Pat. No. 5,109,112), FKBP-12.6 (U.S. Pat. No. 5,457,182), FKBP-13(U.S. Pat. No. 5,498,597), FKBP-14.6 (U.S. Pat. No. 5,354,845), FKBP-52(U.S. Pat. No. 5,763,590), FKBP-56 and FKBP-80, the patents which areincorporated herein by reference.

[0033] The term “isolated” as used herein to refer to a compound of thepresent invention, means altered “by human intervention from its naturalstate. For example, if the compound occurs in nature, it has beenchanged or removed from its original environment, or both. In otherwords, a compound naturally present in a living organism is not“isolated,” but the same compound separated from the coexistingmaterials of its natural state is “isolated”. However, with respect tocompounds found in nature, the compound is isolated if that compound issubstantially free of the materials with which that compound isassociated in its natural state.

[0034] The term “purified” as it refers to a compound means that thecompound is in a preparation that is substantially free of contaminatingor undesired materials. The term purified can also mean that thecompound forms a major component of the preparation, such asconstituting about 50%, about 60%, about 70%, about 80%, about 90%,about 95% or more by weight of the components in the preparation.

[0035] The term “subject” as used herein, refers to an animal,preferably a mammal, who has been the object of treatment, observationor experiment and most preferably a human who has been the object oftreatment and/or observation.

[0036] The term “therapeutically effective amount” as used herein, meansthat amount of active compound or pharmaceutical agent that elicits thebiological or medicinal response in a tissue system, animal or humanthat is being sought by a researcher, veterinarian, medical doctor orother clinician, which includes alleviation of the symptoms of thedisease or disorder being treated.

[0037] The term “composition” is intended to encompass a productcomprising the specified ingredients in the specified amounts, as wellas any product that results, directly or indirectly, from combinationsof the specified ingredients in the specified amounts.

[0038] The term “pharmaceutically acceptable salt” is a salt of one ormore of the inventive compounds. Suitable pharmaceuticallyacceptable-salts of the compounds include acid addition salts which may,for example, be formed by mixing a solution of the compound with asolution of a pharmaceutically acceptable acid such as hydrochloricacid, sulfuric acid, fumaric acid, maleic acid, succinic acid, aceticacid, benzoic acid, citric acid, tartaric acid, carbonic acid orphosphoric acid. Furthermore, where the compounds of the invention carryan acidic moiety, suitable pharmaceutically acceptable salts thereof mayinclude alkali metal salts (e.g., sodium or potassium salts); alkalineearth metal salts (e.g., calcium or magnesium salts); and salts formedwith suitable organic ligands (e.g., ammonium, quaternary ammonium andamine cations formed using counteranions such as halide, hydroxide,carboxylate, sulfate, phosphate, nitrate, alkyl sulfonate and arylsulfonate). Illustrative examples of pharmaceutically acceptable saltsinclude but are not limited to: acetate, adipate, alginate, ascorbate,aspartate, benzenesulfonate, benzoate, bicarbonate, bisulfate,bitartrate, borate, bromide, butyrate, calcium edetate, camphorate,camphorsulfonate, camsylate, carbonate, chloride, citrate, clavulanate,cyclopentanepropionate, digluconate, dihydrochloride, dodecyl sulfate,edetate, edisylate, estolate, esylate, ethanesulfonate, formate,fumarate, gluceptate, glucoheptonate, gluconate, glutamate,glycerophosphate, glycolylarsanilate, hemisulfate, heptanoate,hexanoate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride,hydroiodide, 2-hydroxy-ethanesulfonate, hydroxynaphthoate, iodide,isothionate, lactate, lactobionate, laurate, lauryl sulfate, malate,maleate, malonate, mandelate, mesylate, methanesulfonate, methylsulfate,mucate, 2-naphthalenesulfonate, napsylate, nicotinate, nitrate,N-methylglucamine ammonium salt, oleate, oxalate, pamoate (embonate),palmitate, pantothenate, pectinate, persulfate, 3-phenylpropionate,phosphate/diphosphate, picrate, pivalate, polygalacturonate, propionate,salicylate, stearate, sulfate, subacetate, succinate, tannate, tartrate,teoclate, tosylate, triethiodide, undecanoate, valerate, and the like.

[0039] The term “pharmaceutically acceptable carrier” is a medium thatis used to prepare a desired dosage form of the inventive compound. Apharmaceutically acceptable carrier includes solvents, diluents, orother liquid vehicle; dispersion or suspension aids; surface activeagents; isotonic agents; thickening or emulsifying agents,preservatives; solid binders; lubricants and the like. Remington'sPharmaceutical Sciences, Fifteenth Edition, E. W. Martin (MackPublishing Co., Easton, Pa., 1975) and Handbook of PharmaceuticalExcipients, Third Edition, A. H. Kibbe, ed. (Amer. Pharmaceutical Assoc.2000), both of which are incorporated herein by reference in theirentireties, disclose various carriers used in formulating pharmaceuticalcompositions and known techniques for the preparation thereof.

[0040] The term “pharmaceutically acceptable ester” is an ester thathydrolzyes in vivo into a compound of the present invention or a saltthereof. Illustrative examples of suitable ester groups include, forexample, those derived from pharmaceutically acceptable aliphaticcarboxylic acids such as formates, acetates, propionates, butyrates,acrylates, and ethylsuccinates.

[0041] Compounds of the Present Invention

[0042] In general, the compounds of the present invention are cyclicpolyketides (also referred to as a “macrolides” or “macrolactones”) thatinclude

[0043] as part of their structure and bind to a FKBP wherein R⁴ and R⁵are each selected from the group consisting of hydrogen, methyl, ethyl,and methoxy, provided that at least one of R⁴ and R⁵ is hydrogen,methyl, or ethyl. In preferred embodiments, the FKBP is FKBP-12 orFKBP-52. In more preferred embodiments, the FKBP is FKBP-12 and thecompound binds to FKBP-12 with a K_(d) that is approximately equal to orless than about 100 nM. Compounds of the present invention that bind toFKBP-12 with a K_(d) in the low nanomolar range (e.g., 50 nM, 25 nM, 10nM, 5 nM, 1 nM, and 0.1 nM) are even more preferred.

[0044] In one aspect of the present invention, compounds are of theformula

[0045] are provided wherein:

[0046] R is hydroxyl;

[0047] R¹ is selected from the group consisting of hydrogen, methyl,propyl, ethyl and allyl;

[0048] R² and R³ are each independently hydrogen or hydroxyl;

[0049] R⁴ and R⁵ are each independently selected from the groupconsisting of hydrogen, hydroxyl, methyl, ethyl, and methoxy, providedthat at least one of R⁴ and R⁵ is hydrogen, methyl, or ethyl;

[0050] R⁶ is selected from a group consisting

[0051] wherein

[0052] R⁷ is selected from the group consisting of hydrogen, hydroxyl,methyl, ethyl, and methoxy and R⁸ is selected from a group consisting ofhydrogen, hydroxyl, C₁-C₁₀ alkyl, C₁-C₁₀ alkoxy, aryl, aryloxy,arylalkyl, and arylalkoxy; and,

[0053] a double bond exists between carbon-19 and carbon-20, or

[0054] a double bond exists between carbon-18 and carbon-19 and R and R²together are oxygen forming a lactone ring.

[0055] In one embodiment, the compounds are of formula I or II where R¹,R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ are as previously defined provided thatFK-506, FK-520, 13-desmethoxy-FK-520, and13-desmethoxy-13-methyl-FK-520, 18-hydroxy-FK-520, and 18-hydroxy-FK-506are excluded.

[0056] In another aspect of the invention, compounds of the formula:

[0057] are provided wherein:

[0058] R is hydroxyl;

[0059] R¹ is selected from the group consisting of hydrogen, methyl,ethyl, and allyl;

[0060] R² and R³ are each independently hydrogen or hydroxyl;

[0061] R⁴ and R⁵ are each independently selected from the groupconsisting of hydrogen, hydroxyl, methyl, ethyl, and methoxy, providedthat at least one of R⁴ and R⁵ is hydrogen, methyl, or ethyl;

[0062] R⁷ and R⁸ are each independently selected from a group consistingof hydrogen, hydroxyl, C₁-C₁₀ alkyl, C₁-C₁₀ alkoxy, aryl, aryloxy,arylalkyl, and arylalkoxy; and,

[0063] a double bond exists between carbon-19 and carbon-20, or

[0064] a double bond exists between carbon-18 and carbon-19 and R and R²together are oxygen forming a lactone ring.

[0065] In one embodiment, the compounds are of formula III wherein

[0066] R is hydroxyl;

[0067] R¹ is selected from the group consisting of hydrogen, methyl,ethyl, and allyl;

[0068] R² and R³ are each independently hydrogen or hydroxyl;

[0069] R⁴ and R⁵ are each independently selected from the groupconsisting of hydrogen, hydroxyl, methyl, ethyl, and methoxy, providedthat at least one of R⁴ and R⁵ is hydrogen, methyl, or ethyl;

[0070] R⁷ is selected from the group consisting of hydrogen, hydroxyl,methyl, ethyl, C₁-C₅ alkoxy and aryloxy;

[0071] R⁸ is selected from a group consisting of hydrogen, hydroxyl,

[0072] wherein

[0073] R⁹ is selected from the group consisting of hydrogen, hydroxyl,halide, C₁-C₅ alkyl, C₁-C₅ hydroxyalkyl, and C₁-C₅ alkoxy; and,

[0074] a double bond exists between carbon-19 and carbon-20, or

[0075] a double bond exists between carbon-18 and carbon-19 and R and R²together are oxygen forming a lactone ring.

[0076] In another aspect of the present invention, the compounds of theformula

[0077] are provided wherein

[0078] R¹ is ethyl or allyl;

[0079] R³ is hydrogen or hydroxyl;

[0080] R⁴, R⁵ and R⁷ are each independently selected from the groupconsisting of hydrogen, hydroxyl, methyl, ethyl, and methoxy; and

[0081] R⁸ is selected from a group consisting of hydrogen, hydroxyl,C₁-C₅ alkoxy, and heteroaryloxy, provided that at least one of R⁴ and R⁵is hydrogen, methyl or ethyl.

[0082] In one embodiment, the compounds are of formula IV wherein

[0083] R¹ is ethyl;

[0084] R³ is hydrogen or hydroxyl;

[0085] R⁴ and R⁵ are each independently selected from the groupconsisting of hydrogen, hydroxyl, methyl, ethyl, and methoxy;

[0086] R⁷ is methoxy; and,

[0087] R⁸ is hydroxyl.

[0088] In another embodiment, the compounds are of formula IV wherein

[0089] R¹ is ethyl;

[0090] R³ is hydrogen or hydroxyl;

[0091] R⁴ is selected from the group consisting of hydrogen, hydroxyl,methyl, and ethyl;

[0092] R⁵ is methoxy;

[0093] R⁷ is methoxy; and,

[0094] R⁸ is hydroxyl.

[0095] In another embodiment, the compounds are of formula IV wherein

[0096] R¹ is ethyl;

[0097] R³ is hydrogen or hydroxyl;

[0098] R⁴ is selected from the group consisting of hydrogen, hydroxyl,methyl, and ethyl;

[0099] R⁵ is ethyl;

[0100] R⁷ is methoxy; and,

[0101] R⁸ is hydroxyl.

[0102] In another aspect of the present invention, compounds of theformula

[0103] are provided wherein

[0104] R¹ is ethyl or allyl; and

[0105] R⁴ and R⁵ are each independently selected from the groupconsisting of hydrogen, hydroxyl, methyl, ethyl, and methoxy; providedthat at least one of R⁴ and R⁵ is hydrogen, methyl or ethyl. Thesecompounds are particularly preferred for use as immunosuppressants.Examples of compounds of formulas V-A and V-B include but are notlimited to those listed in Table 1. TABLE 1 Formula R¹ R⁴ R⁵ V-A or V-Ballyl methoxy hydrogen V-A or V-B allyl methoxy methyl V-A or V-B allylmethoxy ethyl V-A or V-B allyl hydrogen hydrogen V-A or V-B allylhydrogen methyl V-A or V-B allyl hydrogen ethyl V-A or V-B allylhydrogen methoxy V-A or V-B allyl methyl hydrogen V-A or V-B allylmethyl methyl V-A or V-B allyl methyl ethyl V-A or V-B allyl methylmethoxy V-A or V-B ethyl methoxy hydrogen V-A or V-B ethyl methoxymethyl V-A or V-B ethyl methoxy ethyl V-A or V-B ethyl hydrogen hydrogenV-A or V-B ethyl hydrogen methyl V-A or V-B ethyl hydrogen ethyl V-A orV-B ethyl hydrogen methoxy V-A or V-B ethyl methyl hydrogen V-A or V-Bethyl methyl methyl V-A or V-B ethyl methyl ethyl V-A or V-B ethylmethyl methoxy

[0106] In another aspect of the present invention, compounds havingtargeted specificity are provided. In one embodiment, compounds of theformula

[0107] are provided wherein

[0108] R¹ is ethyl or allyl;

[0109] R⁴ and R⁵ are each independently selected from the groupconsisting of hydrogen, hydroxyl, methyl, ethyl, and methoxy; providedthat at least one of R⁴ and R⁵ is hydrogen, methyl or ethyl. Thesecompounds possess neurotrophic activity without also possessingimmunosuppressive activity so are particularly preferred for use asneurotrophic agents. Examples of compounds of formulas VI-A and VI-Binclude but are not limited to those listed in Table 2. TABLE 2 FormulaR¹ R⁴ R⁵ VI-A or VI-B allyl methoxy hydrogen VI-A or VI-B allyl methoxymethyl VI-A or VI-B allyl methoxy ethyl VI-A or VI-B allyl hydrogenhydrogen VI-A or VI-B allyl hydrogen methyl VI-A or VI-B allyl hydrogenethyl VI-A or VI-B allyl hydrogen methoxy VI-A or VI-B allyl methylhydrogen VI-A or VI-B allyl methyl methyl VI-A or VI-B allyl methylethyl VI-A or VI-B allyl methyl methoxy VI-A or VI-B ethyl methoxyhydrogen VI-A or VI-B ethyl methoxy methyl VI-A or VI-B ethyl methoxyethyl VI-A or VI-B ethyl hydrogen hydrogen VI-A or VI-B ethyl hydrogenmethyl VI-A or VI-B ethyl hydrogen ethyl VI-A or VI-B ethyl hydrogenmethoxy VI-A or VI-B ethyl methyl hydrogen VI-A or VI-B ethyl methylmethyl VI-A or VI-B ethyl methyl ethyl VI-A or VI-B ethyl methyl methoxy

[0110] In another aspect of the present invention, compounds of theformula VI-A are provided wherein

[0111] R¹ is ethyl;

[0112] R⁴ is methoxy; and,

[0113] R⁵ is selected from the group consisting of hydrogen, hydroxyl,methyl, and ethyl.

[0114] In another aspect of the present invention, compounds of theformula VI-A are provided wherein

[0115] R¹ is ethyl;

[0116] R⁴ is ethyl; and,

[0117] R⁵ is selected from the group consisting of hydrogen, hydroxyl,methyl, and ethyl.

[0118] Because all of the above described FK-like compounds lack amethoxy group at carbon 13 and/or carbon 15 and/or carbon 31 (which arereferred to as C-13 desmethoxy, C-15 desmethoxy, and C-31 desmethoxyrespectively), the initial demethylation reactions to whichFK-506/FK-520 are subjected by one or more P450 enzymes do not occur,thus modulating the normal FK-506/FK-520 metabolism.

[0119] Methods for Making the Inventive Compounds

[0120] The compounds of the present invention can be made, for example,by the genetic manipulation of the FK-520 or FK-506 polyketide synthase(“PKS”) gene in a FK-520 or a FK-506 producing host cell alone or incombination with subsequent chemical modification of the compounds.

[0121] The nucleotide sequence of the FK-520 PKS gene from Streptomyceshygroscopicus var. ascomycetiucus (ATCC 14891) has been deposited inGenBank and assigned Accession No. AF235504. Cosmids, pKOS034-124 (ATCCPTA-729), pKOS034-120 (ATCC PTA-728), pKOS065-M27 (ATCC PTA-726), andpKOS065-M21 (ATCC PTA-727) containing overlapping fragments of theFK-520 PKS gene have been deposited with the American Type CultureCollection, 10801 University Blvd., Manassas Va., 20110-2209 USA(“ATCC”) on Sep. 20, 1999.

[0122] FK-520 derivatives are generally made by the expression of theseand modified constructs in suitable host cells such as those thatnormally produce FK-520. FK-506 derivatives may be made analogously.Alternatively, the recombinant PKS constructs can be expressed in aheterologous host cell as described in U.S. Pat. Nos. 5,672,491 and6,033,883 and PCT publication No. WO 99/02699, each of which isincorporated herein by reference.

[0123] For the purposes of illustration, an abbreviated description isprovided for making novel recombinant PKS constructs using the FK-520PKS gene. The FK-520 PKS enzyme is composed of the fkbA, fkbB, fkbC, andfkbP gene products and synthesizes the core structure of the FK-520molecule. The fkbB open reading frame encodes the loading module and thefirst four extender modules of the PKS. The fkbC open reading frameencodes extender modules five and six of the PKS. The fkbA open readingframe encodes extender modules seven, eight, nine, and ten of the PKS.The fkbP open reading frame encodes the non-ribosomal peptide synthetaseof the PKS that attaches the pipecolic acid moiety and cyclizes theresulting polyketide.

[0124] The polyketide product of the PK-520 PKS is subjected to severalpost-synthetic modifications to form FK-520. The keto group at C-9 isformed from a hydroxylation reaction mediated by the fkbD gene product,a P450 hydroxylase, followed by an oxidation reaction mediated by thefkbO gene product. The C-31 methoxy group is formed from a methylationreaction that is mediated by the fkbM gene product, anO-methyltransferase. The C-13 and C-15 methoxy groups are believed to beformed by methylation reactions by a methyltransferase that is believedto be encoded by the fkbG gene. This methyltransferase is believed toact on the hydroxymalonyl CoA substrates prior to binding of thesubstrate to the AT domains of the PKS during polyketide synthesis.

[0125] Any of fkbA, fkbB, fkbC, fkbP, fkbD, fkbG, fkbM and fkbO geneseither individually or collectively may be modified in the practice ofthe present invention to make novel FK-derivatives. The modificationsmay be in the genes that encode the PKS or in the genes that encode oneor more of the tailoring enzymes. An illustration of a modification in atailoring enzyme is, for-example, deletion of the methyltransferaseactivity encoded by the fkbM gene to yield a compound of the inventionhaving a hydroxyl group at C-31.

[0126] Other modifications include alterations in the specificity and/oractivity of one or more domains that comprise the PKS. In anillustrative embodiment, the AT domain of module 4 is replaced with amalonyl specific AT domain to provide a PKS that produces21-desethyl-FK520, or is replaced with a methylmalonyl specific ATdomain to provide a PKS that produces 21-desethyl-21-methyl-FK520.

[0127] In another illustrative embodiment, the KR and DH codingsequences of module 5 are replaced with those encoding only a KR domainfrom-another PKS gene. The resulting PKS genes code for the expressionof an FK-520 PKS that produces an FK-520 analog that lacks the C-19 toC-20 double bond of FK-520 and has a C-20 hydroxyl group. Alternatively,the DH domain of module 5 may be deleted or otherwise rendered inactive.

[0128] In another example, the coding sequences for extender module sixis replaced with those for an extender module having a methylmalonylspecific AT and only a KR domain from a heterologous PKS gene, such as,for example, the coding sequences for extender module two encoded by theeryAI gene. The resulting PKS genes code for the expression of an FK-520PKS that produces an FK-520 analog that has a C-18 hydroxyl group.Alternatively, the DH and ER domains of module 6 may be deleted orotherwise rendered inactive.

[0129] In another illustrative embodiment, the AT domain of module 7,which specifies a methoxymalonyl CoA and from which the C-15 methoxygroup of FK-520 is derived is replaced by an AT domain that specifies amalonyl, methylmalonyl or ethylmalonyl CoA. Examples of such replacementAT domains include the AT domains from modules 2, 3, and 14 of therapamycin PKS, modules 1 and 2 of the erythromycin (DEBS) PKS, andmodule 4 of the FK-520 PKS. Constructs where module 7 is replaced withan AT domain from extender module 2 or extender module 14 of therapamycin PKS result in a PKS that produces 15-desmethoxy FK-520(hydrogen at C-15). Constructs with an AT domain from extender module 3from the rapamycin PKS or from extender modules 1 or 2 of theerythromycin (DEBS) PKS provide a PKS that produces15-desmethoxy-15-methyl-FK520. Constructs with an AT domain fromextender module 4 of the FK-520 PKS provide a PKS that produces15-desmethoxy-15-ethyl FK-520.

[0130] In another example, the AT domain of module 8, which specifies ahydroxymalonyl CoA and from which the C-13 methoxy group of FK-520 isderived, is replaced by an AT domain that specifies a malonyl,methylmalonyl, or ethylmalonyl CoA. Examples of such replacement ATdomains include the AT domains from modules 3, 12, and 13 of therapamycin PKS and from modules 1 and 2 of the erythromycin PKS. Forexample, strain KOS60-135 is derived from Streptomyces hygroscopicus(ATCC 14891) and expresses a recombinant FK-520 PKS in which the ATdomain of extender module 8 has been replaced by the AT domain ofextender module 3 of the rapamycin PKS. KOS60-135 produces13-desmethoxy-13-methyl-FK520. Similarly, strain KOS45-170 is derivedfrom Streptomyces hygroscopicus (ATCC 14891) and expresses a recombinantFK-520 PKS in which the AT domain of extender module 8 has been replacedby the AT domain of extender module 12 of the rapamycin PKS. KOS45-170produces 13-desmethoxy-FK-520.

[0131] In addition to the above, the desired stereochemistry of aparticular two carbon unit may be achieved by replacing its module (orone or more domains of the module) with another module (or one or moredomains of the module) having the desired stereochemistry

[0132] Instead of modifying the individual modules of fkbA, fkbB, andfkbC, the entire gene may be replaced by another. Preferably, suchhybrid PKS enzymes are produced in recombinant Streptomyces host cellsthat produce FK-520 but have been mutated to inactivate the gene whosefunction is to be replaced, for example by the rapamycin PKS gene tomake the hybrid PKS. Particular examples include (i) replacement of therespective fkbC gene with the rapB gene; and (ii) replacement of therespective fkbA gene with the rapC gene. The latter hybrid PKS produces13,15-didesmethoxy-FK-520.

[0133] A number of engineered strains of Streptomyces Hygroscopicus var.ascomycetiucus ATCC 14891 that produce novel compounds of the presentinvention have been deposited with the ATCC or with the AgriculturalResearch Service Culture Collection, 1815 North University Street,Peoria, Ill. 61604 USA (“NRRL”) as summarized in Table 3. TABLE 3Strain^(a) Deposit Site Compound Produced KOS45-170 (PTA-1811) ATCC onMay. 03, 2000 13-desmethoxy-FK-520 KOS60-135 (PTA-1810) ATCC on May. 03,2000 13-desmethoxy-13-methyl-FK-520 KOS132-188 NRRL on Apr. 17, 200113,15-bisdesmethoxy-FK-520 KOS132-191 NRRL on Apr. 17, 2001 13,15-bisdesmethoxy-15-methyl-FK-520 KOS156-25 NRRL on Apr. 17, 200113,15-bisdesmethoxy-15-ethyl-FK-520 KOS156-9A NRRL on Apr. 17, 200115-desmethoxy-FK-520 KOS156-9B NRRL on Apr. 17, 200115-desmethoxy-15-methyl-FK-520 KOS156-26 NRRL on Apr. 17, 200115-desmethoxy-15-ethyl-FK-5201 KOS156-33A NRRL on Apr. 17, 200113,15-bismethoxy-13-methyl-FK-520 KOS156-33B NRRL on Apr. 17, 200113,15-bismethoxy-13-methyl-15-methyl-FK-520 KOS156-33C NRRL on Apr. 17,2001 13,15-bismethoxy-13-methyl-15-ethyl-FK-520

[0134] These strains and the compounds they produced are embodiments ofthe present invention.

[0135] Methods for making these and other host cells of the inventionthat produces bioengineered FK-520 derivatives and FK-506 derivativesare also described in U.S. Ser. No. 09/410,551 filed Oct. 1, 1999 byinventors Christopher Reeves, Daniel Chu, Chaitan Khosla, Daniel Santi,and Kai Wu entitled POLYKETIDE SYNTHASE ENZYMES AND RECOMBINANT DNACONSTRUCTS THEREFOR which is incorporated herein by reference.

[0136] Host cells can be grown and fermented and the novelFK-derivatives they produce can be isolated and purified from thefermentation broth of these cells using standard procedures. Example 2describes a fermentation method for growing host cells using trypic soybroth with reference to the fermentation of KOS45-170 (which produces13-desmethoxy-FK-520). Example 3 describes an alternate fermentationmethod with reference to KOS60-135 (which produces13-desmethoxy-13-methyl-FK-520). Other host cells of the invention maybe grown using either method by substituting the desired host cell forKOS45-170 in Example 2 or for KOS60-130 in Example 3.

[0137] Examples 4 and 5 describe the purification and characterizationof 13-desmethoxy-FK-520 from the fermentation of KOS45-170. Examples 6and 7 describe the purification of 13-desmethoxy-13-methyl-FK-520 fromthe fermentation of KOS60-135. See also, U.S. Pat. Nos. 5,194,378;5,116,756; and 5,494,820, each of which is incorporated herein byreference. Example 8 describes a general purification protocol fromfermentation and summarizes the ¹³C-NMR data for select compounds of thepresent invention.

[0138] Optional Chemical Derivation

[0139] Once the FK-derivatives of the present invention have beenisolated, they may be further modified using synthetic methods. See e.g.Advanced Organic Chemistry 3rd Ed. by Jerry March (1985) which isincorporated herein by reference.

[0140] For example, although compounds having a hydroxyl at C-18 can bemade genetically by altering module six or fkbC of the FK-520 or FK-506PKS gene, they can also be made by subsequent chemical modification. Aparticularly effective selective hydroxylation can be achieved at C-18by using a general selenium dioxide protocol described by Umbreit andSharpless, 1977, JACS 99(16): 1526-28 that has been modified for usewith FK-like compounds. The procedure generally involves an ene reactionwith selenic acid followed by a [2,3] sigmatropic rearrangement.Briefly, about 1 equivalent of an inventive compound is reacted withabout 1.5 equivalents of SeO₂ and about 7 equivalents of t-BuOOH,preferably in the presence of some water, to yield the corresponding18-hydroxy-FK-derivative. Example 9 describes the direct hydroxylationmethod in greater detail with reference to the C-18 hydroxylation of13-desmethoxy-13-methyl-FK-520. The 18-ene, 20 oxa derivatives are madeby treating an optionally protected 18-hydroxy compound with acid.Example 10 describes this method in greater detail with reference thesynthesis of 18-ene-20-oxa-13-desmethoxy-13-methyl-FK-520.

[0141] Chemical modifications can also be made at C-32. In one method, ametal halogen exchange reaction is used where a halogenated compound(“ZX” wherein X is a halogen) is reacted with for example, nBuli, toform the corresponding lithiated compound, ZLi. The lithiated halogencompound (ZLi) becomes ligands for bismuth upon reacting with BiCl₃(yielding BiZ₃). The bismuthane resulting from reaction of BiZ₃ withbenzoyl peroxide is reacted with a compound of interest to yield thecorresponding FK-derivative with Z at the C-32 position. Scheme 1 is aschematic illustration of one embodiment of this method wheretris[1-(2-t-butyldimethylsilyloxyethyl)indol-5-yl]bismuthane is used tomake 32-[1-(2-hydroxyethyl)-indol-5-yl]-compounds.

[0142] Detailed protocols are found in the Examples. Example 10describes an illustrative protocol for making1-(2-hydroxyethyl)-5-bromoindole from 5-bromoindole and for making thecorresponding bismuthane. Examples 11 and 12 describe the use oftris[1-(2-t-butyldimethylsilyloxyethyl)indol-5-yl]bismuthane to make32-[1-(2-Hydroxyethyl)-indol-5-yl]-13-desmethoxy-13-methyl-FK-520 and32-[1-(2-Hydroxyethyl)-indol-5-yl]-13-desmethoxy-FK-520 respectively.

[0143] Another method for making C-32 derivatives involves convertingthe hydroxyl group at this position into a better leaving group and thensubsequently displacing the group with a moiety of interest. Scheme 2describes two illustrative protocols.

[0144] In the first reaction shown in Scheme 2, a compound of thepresent invention is selectively reacted with trifluoromethanesulfonicanhydride in the presence of a base to yield the C-32 O-triflatederivative. SN2-displacement of the triflate with aryl compounds such as1H-tetrazole provides the corresponding C-32 aryl derivative. Thisstrategy is particularly effective when the moiety of interest is a poornucleophile.

[0145] In the second reaction shown in Scheme 2, the hydroxyl at C-32 isdisplaced with a good nucleophile. Here, the inventive compound isreacted with p-nitrophenylchloroformate to yield the correspondingcarbonate. The p-nitrophenol is subsequently displaced with an aminocompound to provide the corresponding carbamate derivative.

[0146] Other chemical modifications include C-32-O-aralkyl ethers. Thesecompounds can be made by following the procedure described by Goulet etal., 1998, Bioorg. Med. Chem. Lett. 8: 2253-2258, which is incorporatedherein by reference. Derivatives with a ═N—NH(C═O)NH₂ moiety at C-32 canbe made using a protocol described by Example 19 of U.S. Pat. No.5,604,294, which is incorporated herein by reference. U.S. Pat. Nos.4,894,366; 5,247,076; 5,252,732; 5,349,061; 5,457,111; 5,877,184 and6,504,294 describe making additional modifications at the cyclohexylring and are also incorporated herein by reference. Chemicalmodifications can also be made at the C-31 position in compoundspossessing a C-31 hydroxyl that are similar to those described for theC-32 hydroxyl. Modifications where the cyclohexyl ring is replaced withother moieties are described by U.S. Pat. No. 5,612,350 which is alsoincorporated herein by reference.

[0147] Formulation

[0148] A composition of the present invention generally comprises one ormore compound of the present invention and a pharmaceutically acceptablecarrier. The inventive compound may be in free form or where appropriateas pharmaceutically acceptable derivatives such as prodrugs, and saltsand esters of the inventive compound.

[0149] The one or more compounds of the present invention are includedin the pharmaceutical composition in an amount sufficient to produce thedesired effect upon the disease process or condition. In preferredembodiments, the amount of active ingredient may range between about0.01 mg to 50 mg and more preferably between about 0.1 mg to 10 mg. Ineven more preferred embodiments, the amount of active ingredients rangefrom about 0.5 mg to about 5 mg. Convenient dosages amounts include 0.5mg, 1 mg and 5 mg units.

[0150] The composition may be in any suitable form such as solid,semisolid, or liquid form (e.g., tablets, pellets, capsules,suppositories, solutions, emulsions, suspensions, etc.). SeePharmaceutical Dosage Forms and Drug Delivery Systems, 5^(th) edition,Lippicott Williams & Wilkins (1991) which is incorporated herein byreference. In general, the pharmaceutical preparation will contain oneor more of the compounds of the invention as an active ingredient inadmixture with an organic or inorganic carrier or excipient suitable forexternal, enteral, or parenteral application. The active ingredient maybe compounded, for example, with the usual non-toxic, pharmaceuticallyacceptable carriers for tablets, pellets, capsules, suppositories,pessaries, solutions, emulsions, suspensions, and any other formsuitable for use. The carriers that can be used include water, glucose,lactose, gum acacia, gelatin, mannitol, starch paste, magnesiumtrisilicate, talc, corn starch, keratin, colloidal silica, potatostarch, urea, and other carriers suitable for use in manufacturingpreparations, in solid, semi-solid, or liquefied form. In addition,auxiliary stabilizing, thickening, and coloring agents and perfumes maybe used.

[0151] Where applicable, an inventive compound may be formulated asmicrocapsules and nanoparticles. General protocols are described forexample, by Microcapsules and Nanoparticles in Medicine and Pharmacy byMax Donbrow, ed., CRC Press (1992) and by U.S. Pat. Nos. 5,510,118;5,534,270; and 5,662,883 which are all incorporated herein by reference.By increasing the ratio of surface area to volume, these formulationsallow for the oral delivery of compounds that would not otherwise beamenable to oral delivery.

[0152] An inventive compound may also be formulated using other methodsthat have been previously used for low solubility drugs. For example,the compounds may form emulsions with vitamin E or a PEGylatedderivative thereof as described by PCT Publications WO 98/30205 and WO00/71163 which are incorporated herein by reference. Typically, theinventive compound is dissolved in an aqueous solution containingethanol (preferably less than 1% w/v). Vitamin E or a PEGylated-vitaminE is added. The ethanol is then removed to form a pre-emulsion that canbe formulated for intravenous or oral routes of administration. Anotherstrategy involves encapsulating the inventive compounds in liposomes.

[0153] Yet another method involves formulating an inventive compoundusing polymers such as polymers such as biopolymers or biocompatible(synthetic or naturally occurring) polymers. Biocompatible polymers canbe categorized as biodegradable and non-biodegradable. Biodegradablepolymers degrade in vivo as a function of chemical composition, methodof manufacture, and. implant structure. Illustrative examples ofsynthetic polymers include polyanhydrides, polyhydroxyacids such aspolylactic acid, polyglycolic acids and copolymers thereof, polyesterspolyamides polyorthoesters and some polyphosphazenes. Illustrativeexamples of naturally occurring polymers include proteins andpolysaccharides such as collagen, hyaluronic acid, albumin, and gelatin.

[0154] Another method involves conjugating a compound of the presentinvention to a polymer that enhances aqueous solubility. A particularlyeffective method involves conjugating polyethylene glycol or apoly-amino acid such as poly-glutamic acid or poly-aspartic acid viaester linkages to one or more hydroxyl groups (e.g. such as off carbons10, 24 and where applicable, off carbon 18) of the compound.Illustrative examples of suitable polymers include polyethylene glycol,poly-(d-glutamic acid), poly-(1-glutamic acid), poly-(1-glutamic acid),poly-(d-aspartic acid), poly-(1-aspartic acid), poly-(1-aspartic acid)and copolymers thereof. Polyethylene glycol conjugated compounds canalso be made as essentially described by U.S. Pat. No. 5,922,729 whichis incorporated herein by reference. Poly-amino acid conjugatedcompounds, particularly poly-glutamic acid conjugated compounds may beprepared as described by U.S. Pat. No. 5,977,163 which is incorporatedherein by reference.

[0155] In another method, an inventive compound is conjugated to amonoclonal antibody. This strategy allows the targeting of the inventivecompound to specific targets. General protocols for the design and useof conjugated antibodies are described in Monoclonal Antibody-BasedTherapy of Cancer by Michael L. Grossbard, ed. (1998), which isincorporated herein by reference.

[0156] In addition, specific formulations previously described forFK-506 can be adapted for use with the inventive compounds. For example,U.S. Pat. No. 5,955,469 and PCT Publication No. WO 99/49863, which areincorporated herein by reference, provide methods for making emulsionsfor all applications including oral and intravenous use. U.S. Pat. Nos.5,939,427 and 5,385,907, and PCT Publication Nos. 96/13249, 99/24036,and 00/32234, which are also incorporated herein by reference, describelotion and ointment formulations.

[0157] U.S. Pat. Nos. 5,338,684 and 5,260,301 describe solutionformulations for intravenous use and for injections. Briefly, due to therelatively poor solubility of the inventive compounds in water, thecompounds of the present invention are admixed with a castor-oil typesurface active agent such as HCO (polyoxyethylated castor oil), mostpreferably HCO-60 (trademark, prepared by Nikko Chemicals Co.), and/oradmixed with an organic solvent, most preferably ethanol. As a result,formulations may comprise a compound of the present invention andpolyoxyethylated castor oil and/or ethanol. An illustrative example ofsuch a formulation comprises 5 mg of anhydrous Compound, 200 mg ofpolyoxyl 60 hydrogenated castor oil (HCO-60) and dehydrated ethanol USP,80% v/v. This formulation can be packaged in 1 mL single dose ampulesand can optionally be diluted with 0.9% sodium chloride or 5% dextrosesolution prior to intravenous use.

[0158] Methods of Treating Patients

[0159] The compounds of the present invention are useful in treatingdisease conditions as described for FK-506 (also known as tacrolimus) inU.S. Pat. Nos. 5,955,469; 5,542,436; 5,365,948; 5,348,966; and5,196,437, incorporated herein by reference. In one embodiment, theinventive compounds and compositions are used as immunosuppressiveagents. In another embodiment, the inventive compounds and compositionsare used as neurotrophic agents. In yet another embodiment, theinventive compounds and composition are used as agents to treatanti-inflammatory disorders, particularly inflammatory skin diseasessuch as psoriasis and dermatitis. The method generally comprisesadministering a therapeutically effective amount of an inventivecompound to a subject in need thereof.

[0160] The compounds of the present invention are administered on anas-need basis and may be given to patients continuously or anintermittent basis, such as hourly, semi-daily, daily, semi-weekly,weekly, semi-monthly, or monthly intervals. In general, the dosage isthe minimum amount of compound that is needed to effectuate the desiredeffect.

[0161] When the Compound is taken internally, a useful marker ofdetermining the appropriate dosage is the whole blood troughconcentration which should generally range from 0.01 picomole of drugper 1 mL of whole blood (1 picomole/mL) to 50 picomole of drug per 1 mlof whole blood (0.01 picomole/mL). In preferred embodiments, the dosageis the amount required to maintain a whole blood trough concentration ofbetween about 1 picomole/mL and about 30 picomole/mL, and morepreferably between about 10 picomole/mL and about 20 picomole/mL. Molesare used to express the amounts of compound since weight amounts aredependent on the molecular weight of a particular Compound.

[0162] When the compounds of the present invention are used asimmunosuppressants, they may be used in a similar manner as FK-506. Tothat end, although many parameters are expected to be different due tothe metabolic stability of the compounds of the present invention, thepharmacological values for FK-506 provide useful benchmarks forcomparisons and may be found on the internet at the following URL:http://www.fujisawausa.com/medinfo/pi/pi_page_pg.htm which isincorporated herein in its entirety.

[0163] There are a number of advantages of using the compounds of thepresent invention over that of using FK-506. One benefit is that theinventive compounds are metabolized more slowly than FK-506 enablinglower dosages and/or fewer numbers of doses per unit time period. Forexample, if the inventive compound is being administered by continuousintravenous infusion, a lower dosage may be used instead of the amountequivalent (in moles) to 0.03-0.05 mg/kg/day that is recommended forFK-506. If the inventive compound is being administered orally, thenlower dosages may also be used instead of the amount that is equivalentto between about 0.05 and 0.10 mg/kg every twelve hours. Alternatively,the same amount may be used but because the compounds of the presentinvention are metabolized more slowly, they may be administered lessfrequently. For example, the amount that is equivalent to an oral doseof between about 0.05 and 0.10 mg/kg of FK-506 may be administered dailyinstead of every twelve hours. Finally, a combination of lower dose andless frequent administration may be used.

[0164] Another benefit is fewer drug interactions since the Inventivecompounds are not significantly affected by P450 activity. As a result,a larger arsenal of drugs available to treat complications that mayoccur. For example, a common side effect of taking FK-506 ishypertension. Because commonly used anti-hypertensive agents are calciumchannel blockers that modulate P450 activity levels and thus blood levelof FK-506, treating patients with both FK-506 and a calcium channelblocker may be problematic. In contrast, no such problems areanticipated with the compounds of the present invention. If a patientbeing treated with an Inventive compound described herein developshypertension, then that patient may be treated using a method comprisingadministering a compound of the present invention to treat theunderlying ailment and administering an antihypertensive agent. Becausethe compounds are more resistant to P450 mediated metabolism, calciumchannel blocking agents such as diltiazem, nicardipine, nifedipine, andverapamil may be more readily used.

[0165] Other examples of drugs that elevate FK-506 levels but may beused more readily with compounds of the present invention include butare not limited to: antifungal agents (such as clotrimazole,fluconazole, itraconazole, and ketoconazole); macrolide antibiotics(such as clarithromycin, erythromycin, troleandomycin); gastrointestinalprokinetic agents (such as cisapride and metoclopramide); andmiscellaneous drugs such as bromocriptine, cimetidine, cyclosporine,danazol, methylprednisolone, and protease inhibitors. Examples of drugsthat decrease FK-506 levels that may now be used more readily with acompound of the present invention include anticonvulsants (such ascarbamazepine, phenobarbital, phenytoin) and antibiotics (such asrifabutin, and rifampin).

[0166] Use of the compounds of the present invention as neurotrophicagents (including dosing protocols) are generally similar to thoseoutlined above for use as immunosuppressants. The benefits of usingcompounds of the present invention that are more metabolically stableover compounds like FK-506 and FK-520 are identical to that describedabove.

[0167] For the reasons stated above, practice of the present inventionresults in potentially fewer side effects and toxicities from thegenerally lower drug concentrations. Additional benefits includeconvenience for both patient and health care provider. In particular,blood levels of the compounds described herein do not need to bemonitored as carefully or as frequently since individual variations inP450 activity are less important in the metabolism rate of thesecompounds and fewer drug-drug interactions are expected. Consequently, astandardized dosing schedule may be developed that is more generallyapplicable.

[0168] A detailed description of the invention having been providedabove, the following examples are given for the purpose of illustratingthe present invention and shall not be construed as being a limitationon the scope of the invention or claims.

EXAMPLE 1 Recombinant PKS Genes for FK-506 and FK-520 Compounds HavingVariations at C-13 and C-15 Positions

[0169] This Example provides construction protocols for recombinantFK-520 (Streptomyces hygroscopicus (ATCC 14891)) and FK-506 (fromStreptomyces sp. MA6858 (ATCC 55098)), described in U.S. Pat. Nos.5,116,756, incorporated herein by reference) PKS genes in which theextender module 7 and/or extender module 8 AT coding sequences have beenreplaced with an AT domain from another PKS. The AT domains of extendermodule 7 and 8 of FK-520 and FK-506 are believed to specifymethoxymalonyl CoA from which the methoxy groups at both C-13 and C-15are derived. Replacement of the AT domains of extender module 7 and/or 8with an AT domain that specifies other CoA esters such as malonyl CoA,methylmalonyl CoA and ethylmalonyl CoA results in compounds having ahydrogen, methyl, or ethyl at the C-13 and C-15 positions. Table 4summaries the AT domains, their CoA specificities and the resultinggroup at the C-13/C15 positions. TABLE 4 RESULTING AT DOMAIN AT DOMAINORIGIN AT SPECIFICITY GROUP rapAT2 extender module 2 of malonyl CoAhydrogen rapamycin PKS rapAT12 extender module 12 of malonyl CoAhydrogen rapamycin PKS rapAT14 extender module 14 of malonyl CoAhydrogen rapamycin PKS eryAT2 extender module 2 of methyl malonyl CoAmethyl erythromycin (DEBS) PKS rapAT3 extender module 3 of rapamycinmethyl malonyl CoA methyl PKS FK520AT4 extender module 4 of FK- ethylmalonyl CoA ethyl 520 PKS

[0170] Phage vector KC515 (based on the broad host range phage ΦC31) isused to deliver a cassette containing the AT domain to be swapped. Thecassette contains one of the above heterologous AT domain insertedbetween two ca. 1.5 kb fragments of DNA identical to the sequencesflanking AT7 or AT8. The resulting recombinant phage is used totransform the FK-520 or FK-506 producer strains. The transformed strainsare cultured to select and identify desired recombinants produced bydouble crossover homologous recombination to yield the desiredrecombinant cells. See also, Examples 1-5 of U.S. Ser. No. 09/410,551filed Oct. 1, 1999 which is incorporated herein by reference.

[0171] Table 5 includes an illustrative list of recombinant cells thatproduces inventive compounds having hydrogen, methyl or ethyl at theC-13 and/or C15 positions. TABLE 5 TARGET AT REPLACEMENT COMPOUNDPRODUCED IN COMPOUND PRODUCED IN FK- DOMAIN(S) AT DOMAIN(S) FK520PRODUCING HOST 506 PRODUCING HOST AT8 rapAT12 13-desmethoxy-FK-520 13desmethoxy-FK-506 (KOS45-170) AT8 rapAT3 13-desmethoxy-13-methyl-FK-52013-desmethoxy-13-methyl-FK-506 (KOS60-135) AT7 rapAT215-desmethoxy-FK-520 15-desmethoxy-FK-506 (KOS156-9A) AT7 rapAT315-desmethoxy-15-methyl-FK-520 15-desmethoxy-15-methyl-FK-506(KOS156-9B) AT7 FK520AT4 15-desmethoxy-15-ethyl-FK-52015-desmethoxy-15-ethyl-FK-506 (KOS156-26) AT8 rapAT1213,15-bisdesmethoxy-FK-520 13,15-bismethoxy-FK-506 AT7 rapAT2(KOS132-188) AT8 rapAT12 13-desmethoxy-15-desmethoxy-15-13-desmethoxy-15-desmethoxy-15- AT7 rapAT3 methyl-FK-520 methyl-FK-520(KOS132-191) AT8 rapAT12 13-desmethoxy-15-desmethoxy-15-13-desmethoxy-15-desmethoxy-15- AT7 FK520AT4 ethyl-FK-520 ethyl-FK-506(KOS156-25) AT8 rapAT3 13-desmethoxy-13-methyl-15-13-desmethoxy-13-methyl-15- AT7 rapAT14 desmethoxy-FK-520desmethoxy-FK-506 (KOS156-33A) AT8 rapAT3 13,15 bisdesmethoxy-13,15-13,15-bisdesmethoxy-13,15- AT7 eryAT2 bismethyl-FK-520 bismethyl-FK-520(KOS156-33B) AT8 rapAT3 13-desmethoxy-13-methyl-15-13-desmethoxy-13-methyl-15- AT7 FK520AT4 desmethoxy-15-ethyl-FK520desmethoxy-15-ethyl-FK506 (KOS156-33C)

[0172] These strains and the compounds they produced are embodiments ofthe present invention.

EXAMPLE2 Production of 13-desmethoxy-FK-520

[0173] A 1 mL vial of KOS-45-170 working cell bank was thawed and thecontents of the vial were added to 50 mL trypic soy broth in a 250 mLbaffled flask. Trypic soy broth was purchase from Difco and a solutionmade at a concentration of 30 g/L. Prior to sterilization, the pH wasadjusted to 6.0 and this solution was used as Medium 1. For growth inflasks, Medium 1 was supplemented with Prior to sterilization, 21.32 g/Lof MES buffer prior to sterilization. 20 mL/L of 500 g/L glucose(sterile filtered) was added post-sterilization. The flask was placed inan incubator/shaker maintained at 30±1° C. and 175±25 RPM for 48±10hours. The 50 mL culture was then added to a 2.8 L baffled flaskcontaining 500 mL of medium. This flask was incubated in anincubator/shaker at 30±1° C. and 175±25 RPM for 48±10 hours.

[0174] A 10 L fermenter was prepared by sterilizing 10 L of Medium 1 at121° C. for 60 minutes. 0.2 L of sterile filtered 500 g/L glucose wasadded to the 10 L fermenter. After incubation, the 500 ml culture wastransferred to a sterile inoculation bottle and aseptically added to the10 L fermenter. The fermenter was controlled at 30° C., pH 6.0 byaddition of 2.5 N H₂SO₄ and 2.5 N NaOH, dissolved oxygen≧50% airsaturation by agitation rate (600-900 RPM) and air flow rate (2-8 LPM).Foam was controlled by the intermittent addition of a 50% solution ofAntifoam B. Growth in the 10L fermenter continued for 48±10 hours.

[0175] A 1000 L fermenter with a 800 L working volume was prepared bysterilizing 800 L of the Medium 1 at 121° C. for 45 minutes. 16 L ofsterile filtered 500 g/L glucose was added aseptically to the 1000 Lfermenter after autoclaving. Culture from the 10 L fermenter wasaseptically transferred to the 1000 L fermenter. The fermenter wascontrolled at 30° C., pH 6.0 by addition of 2.5-5.0.N H₂SO₄ and 2.5-5.0N NaOH, dissolved oxygen≧50% air saturation by agitation rate (100-200RPM), air flow rate (2-250 LPM), and/or back pressure control (0.2-0.4bar). Foam was controlled by the intermittent addition of a 50% solutionof Antifoam B. Production of 13-desmethoxy-FK520 ceased on day 5 and thefermenter was harvested. The fermentation broth was centrifuged at20,500 g in an Alpha Laval AS-26 centrifuge.

EXAMPLE 3 Production of 13-desmethoxy-13-methyl-FK-520

[0176] A 1 mL vial of KOS-60-135 working cell bank was thawed and thecontents of the vial are added to 50 mL Medium 1 in a 250 mL baffledflask. The flask was placed in an incubator/shaker maintained at 30±1°C. and 175±25 RPM for 48±10 hours. The 50 mL culture was then added to a2.8 L baffled flask containing 500 mL of Medium 1. This flask wasincubated in an incubator/shaker at 30±1° C. and 175±25 RPM for 48±10hours. The 500 mL culture was divided equally among ten 2.8 L baffledflasks each containing 500 mL of Medium 1. All flasks were thenincubated as described previously. Medium 1 is the tryptic soy brothdescribed in Example 2.

[0177] A 150 L fermenter was prepared by sterilizing 100 L of Medium 2at 121° C. for 45 minutes. Medium 2 Component Concentration Corn starch16 g/L Corn dextrin (type III) 10 g/L Soy flour 15 g/L Calcium carbonate 4 g/L Corn steep liquor (50%)  5 g/L Soy oil  6 g/L Sodium chloride 2.5g/L  Ammonium sulfate  1 g/L

[0178] After incubation, all 10 flasks were combined in a 5 L sterileinoculation bottle and aseptically added to a 150 L fermenter. Thefermenter was controlled at 30° C., pH 6.0 by addition of 2.5 N H₂SO₄and 2.5 N NaOH, dissolved oxygen≧50% air saturation by agitation rate(50-600 RPM), air flow rate (10-50 LPM), and/or back pressure control(0.1-0.3 bar). Foam was controlled by the intermittent addition of a 50%solution of Antifoam B.

[0179] A 1000 L fermenter with a 700 L working volume was prepared bysterilizing 700 L of Medium 3 at 121° C. for 45 minutes. Medium 3Component Concentration Corn starch 35 g/L Corn dextrin (type III) 32g/L Soy flour 33 g/L Calcium carbonate  8 g/L Corn steep liquor (50%) 12g/L Soy oil  6 g/L Sodium chloride  7 g/L Ammonium sulfate  2 g/L

[0180] Culture from the 100 L fermenter was aseptically transferred tothe 1000 L fermenter. The fermenter is controlled at 30° C., pH 6.0 byaddition of 2.5-5.0 N H₂SO₄ and 2.5-5.0 N NaOH, dissolved oxygen≧50% airsaturation by agitation rate (150-300 RPM), air flow rate (100-600 LPM),and/or back pressure control (0.1-0.4 bar). Foam was controlled by theintermittent addition of a 50% solution of Antifoam B. Production of13-desmethoxy-13-methyl-FK520 ceases on day 5 and the fermenter washarvested. The fermentation broth was centrifuged at 20,500 g in anAlpha Laval AS-26 centrifuge.

EXAMPLE 4 Purification of 13-desmethoxy-FK-520

[0181] Two sources of 13-desmethoxy-FK-520 was used for the purificationprocedure. The first source was centrifuged fermentation broth (1800 L)that was passed through a Sharples centrifuge (15,000 rpm) at a rate of2 liters per minute. In addition, the whole broth was filtered through aCuno filtration unit (model # 16ZPC40F3T10CT) with 4 filter cartridges(10 μm pore size). The second source was 100% methanol extracted cellpaste. The cell paste was obtained in 6 batches from the Sharplescentrifuge bowl during centrifugation. The methanol extract was filteredthrough a Cuno filtration unit (model # 16ZPC40F3T10CT) containing 4filter cartridges (10 μm pore size). The centrifuged and filtered wholebroth was loaded directly onto an HP20 column. The filtered methanolextract was prepared for load onto the HP20 column by adding deionizedwater to get a final concentration of 50% methanol. The two sourcescombined contained 7.4 g of 13-desmethoxy-FK-520. The 50% methanolsolution was loaded on to the HP 20 column after the whole broth. TheHP20 sorbent was packed in an Amicon P350 Moduline chromatography column(35 cm×20 cm) The HP20 column was loaded at 4 L/min and had backpressureunder 5 psi.

[0182] Following loading, the column was washed with 50% methanol andproduct (13-desmethoxy-FK-520) was eluted with 5 column volumes (100 L)of 100% methanol. The product pool was evaporated using a Buchi rotaryevaporator (R-152).

[0183] The solids from evaporation weighed 2 kilogram. The solids weredissolved in a minimal amount of 100% acetone, filtered and the filtratewas evaporated to dryness. This resulted in 357 grams of solidscontaining 3.4% 13-desmethoxy-FK520 by weight. The evaporated solidswere further extracted with 60% methanol, filtered and the filtrateextracted again with 60% methanol. The 60% methanol extraction yielded147 grams of solids of which 4.3% was 13-desmethoxy-FK-520 by weight.

[0184] The solids from methanol extraction were dissolved in 20 L of 50%addition of methanol then water and loaded onto an HP20SS column (8.9cm×30 cm). The column was washed with 2 column volumes of 55% methanol.13-desmethoxy-FK-520 was eluted with 3 column volumes of 60% methanolthen 3 column volumes of 65% methanol. The best pool of fractions had afinal volume of 9.2 L and contained 24 grams of solids. Product purityfor this intermediate was 23%. Each fraction contains 0.5 column volumeseach, and fractions were pooled to maximize the recovery of13-desmethoxy-FK-520 based on HPLC chromatograms.

[0185] The best pool from HP20SS chromatography was diluted with 10.8 Lof deionized water and loaded onto a 1 L C18 column (8.9 cm×16.5 cm).The C18 column was washed with 3 column volumes of 50% methanol and 6column volumes of 60% methanol. The 13-desmethoxy-FK-520 was eluted with10 column volumes of 70% methanol and 6 column volumes of 80% methanol.After C18 chromatography, fractions 20-36 was determined to be the bestpool and contained 10.8 grams of solids, of which 50% was13-desmethoxy-FK-520. Fraction 19 was the start of the 70% methanolelution.

[0186] The best pool 13-desmethoxy-FK-520 from the 1L C18 chromatographywas evaporated to dryness using the Buchi rotary evaporater (R-152) andextracted with dichloromethane. The extract was filtered and filtrateevaporated using a Buchi evaporator giving 9.9 grams of solids of which48% was 13-desmethoxy-FK-520.

[0187] The solids from dichloromethane extraction was dissolved in 2.5 Lof 50% methanol and loaded onto a 4.8 cm×20 cm C18 bakerbond column at10 ml/min. The column was washed with one column volume of 50% methanol.The 13-desmethoxy-FK-520 was eluted with 6 column volumes of 85%methanol. Fractions 3-5 was determined be the best pool, where fraction1 was the start of the 85% methanol elution. The best pool from thischromatography contained 4.43 grams of 13-desmethoxy-FK-520. The bestpool was again diluted to a final volume of 2L of 50% methanol andloaded onto the same column. The column was again washed with one columnvolume of 50% methanol. The 13-desmethoxy-FK-520 was eluted with 6column volumes of 80% methanol. Fractions 4-11 were combined to make a 2L pool. Overall yield was >65%. Other compounds of the invention thatare produced from the fermentation of engineered host cells may bepurified in a similar manner.

EXAMPLE 5 Characterization of 13-desmethoxy-FK520

[0188] A 100-mg sample of partially purified 13-desmethoxy-FK520(Example 4) was dissolved in 1000 uL of acetonitrile and insolubles wereremoved by centrifugation. The supernatant was purified by preparativeHPLC using the following conditions: column=22×50 mm InertSil ODS-3(MetaChem); flow rate=8.0 mL/min; solvent A=H₂O+0.1% acetic acid,solvent B=CH₃CN+0.1% acetic acid; gradient program: time 0=50% B, time2=gradient to 100% B over 15 minutes, time 20=gradient to 90% B over 1minute. Injections of 50 uL were made, and the separation was monitoredby UV absorption at 240 nm. Two major peaks eluted, one broad peakcorresponding to 13-desmethoxy-FK520 (ca. 15 minutes), and a sharp peakat 17 minutes corresponding to the oxepane rearrangement product.

[0189] The two major fractions were evaporated to dryness under vacuum.The residues were evaporated twice from- acetonitrile to remove tracesof acetic acid, then lyophilized from frozen benzene and dried overnightover KOH pellets under vacuum to yield 45 mg of pure 13-desmethoxy-FK520and 20 mg of pure oxepane. NMR analysis indicated that each compoundexists as a mixture of trans:cis amide rotamers. TABLE 6 ¹³C-NMR data(CDCl₃, 300K, 100 MHz): 13-des(OMe)- FK520 13-des(OMe)-FK520FK520-oxepane Carbon Trans Cis Trans Cis Trans Cis  1 168.71 169.00169.31 169.43 169.84 169.32  2 52.70 56.55 52.15 56.31 51.60  3 26.2027.60 26.64 26.47 25.49  4 20.81 21.10 21.34 20.79 20.91  5 24.48 24.5524.94 24.38 25.20  6 43.87 39.24 44.68 39.24 43.65  8 165.78 164.70165.93 164.77 167.40 167.02  9 192.66 196.13 196.33 195.76 98.24 98.1410 98.64 97.06 98.93 98.36 210.13 209.83 11 33.62 34.57 34.84 34.9443.29 11-Me 15.99 16.20 15.82 16.37 16.81 12 32.53 32.68 35.46 13 73.6273.68 27.18 27.37 13-OMe 56.07 56.29 Missing Missing Missing Missing 1472.22 72.86 73.84 70.54 77.00 15 75.22 82.14 81.55 83.04 15-OMe 56.9657.54 57.89 57.71 57.54 16 35.43 32.94 34.38 32.86 17 26.00 26.32 27.4029.74 17-Me 19.50 20.46 21.79 21.34 21.78 18 48.46 48.66 47.01 48.8348.24 19 138.74 139.62 139.26 138.36 138.83 139.45 19-Me 15.83 15.6817.63 16.11 16.81 20 123.34 123.06 124.53 122.98 123.69 123.69 21 54.9354.67 54.94 54.12 54.52 21a 24.48 24.17 23.63 24.57 23.46 21b 11.6711.67 11.70 11.56 11.67 22 213.44 213.51 211.97 213.28 212.70 211.32 2343.54 43.19 44.94 44.21 44.30 24 69.03 70.04 67.74 69.88 69.61 25 40.3439.76 39.39 39.87 39.10 25-Me 9.78 9.48 9.93 9.41 9.59 26 77.21 77.8781.00 77.24 77.76 27 131.78 132.32 130.50 131.84 131.43 27-Me 14.2314.09 12.91 14.21 13.60 28 129.61 129.70 132.97 129.46 130.44 29 34.9034.90 34.94 34.94 34.90 30 34.74 34.84 34.38 34.48 34.58 31 84.16 84.1684.10 84.10 84.19 31-OMe 56.59 56.59 56.49 56.59 56.45 32 73.54 73.5473.41 73.47 73.48 33 31.20 31.20 31.18 31.18 31.16 34 30.62 30.62 30.4530.63 30.46

EXAMPLE 6 Purification of 13-desmethoxy-13-methyl-FK-520

[0190] The starting material containing 417 mg of13-desmethoxy-13-methyl-FK-520 was obtained from two sources. The firstsource was centrifuged and filtered fermentation broth (900 L). TheSharples centrifuge spun the fermentation broth at 15,000 rpm. Thecentrifuged broth then went through a Cuno depth filter containing four10 um filter cartridges at a rate of 2 L/min. The second source was fromthe 100 L of methanol used for extracting the product from the cellpaste. The cells were extracted by adding 100 L of methanol to the cellpaste and then stirring the solution for 3-4 hours. The resultingsolution was then filtered through the same depth filter alreadycontaining the cell solids. The same 100 L of methanol was thenrecirculated through the filter apparatus for 60 minutes. The methanolin the filter was then expelled via air into a container at 2 L/min. Theresulting methanol from the filter was diluted to a 50% methanolicsolution using water.

[0191] The centrifuged and filtered fermentation broth (900 L) waspassed through 18.3 L of HP20 sorbent packed into an Amicon P350SSModuline 2 chromatography column. At 4 L/min loading, back pressure wasfound to be less than 5 psi. Following loading, the resin was washedwith 200 L of the 50% methanolic solution made from the cell pasteextract at a flow rate of 4 L/min. The 13-desmethoxy-13-methyl-FK-520was eluted using 60 L of 100% methanol at a flow rate of 1 L/min.

[0192] The product pool was evaporated using a Buchi rotary evaporator(R-153). The 175 g of solids were dissolved in 2 L of 100% methanol,filtered and the filtrate evaporated to dryness. After the methanolextraction, 145 g containing 417 mg of 13-desmethoxy-13-methyl-FK-520remained. The solids were extracted twice using 1 L of a 9:1 solution ofhexane:acetone in a 20 L round bottom flask in a 40° C. water bath for30 min. The solution was then filtered and the filtrate was rotovappeddown to dryness. The resulting solids were crushed and then extractedfor 30 minutes in a 20% solution of acetone in hexane in a beaker withvigorous mixing using a Lightning Labmaster mixer with an A310 rotor at1000 rpm. The resulting solution was filtered and the filtrate contained61.5 g of solids containing 0.7% 13-desmethoxy-13-methyl-FK-520 byweight. The filtrate was dried down and then resuspended in 4.10 L of60% methanol.

[0193] The material then went through a 4.8 cm×26 cm Konteschromatography column containing washed and equilibrated C18 sorbent ata rate of 100 ml/min. The 13-desmethoxy-13-methyl-FK520 was then elutedwith 85% methanol at a flow rate of 100 ml/min over 5 CV. Half columnvolume fractions were taken and like fractions were pooled. The poolswere then diluted to 60% methanol and the entire procedure was repeatedfor a total of 4 times. At the end of the 4^(th) C18 chromatographystep, 850 mg of solids containing 49% 13-desmethoxy-13-methyl-FK-520 byweight remained. Overall recovery at this step was 100%. The best poolwas rotovapped down and redissolved in 1.36 L of 60% methanol. Thesolution was then loaded onto a 2.5 cm×56 cm Kontes chromatographycolumn containing 275 ml of washed and equilibrated C18 sorbent at aflow rate of 25 ml/min. The material was then eluted with 85% methanolat the same flow rate over 12 column volumes. Fractions containing13-desmethoxy-13-methyl-FK-520 were pooled and the chromatography wasrepeated once more. The best pool at the end of this step contained 419mg of 13-desmethoxy-13-methyl-FK-520 with >50% purity. The best pool(1.84 L) was diluted to 60% methanol and reloaded onto a 4.8 cm×25 cmKontes chromatography column containing washed and equlibrated C 18sorbent at a rate of 100 ml/min. The run was monitored by UV at 210 nmand a heart cut of the major peak was taken.

[0194] The dried solids were then extracted twice (50 ml each) withhexane, dichloromethane, and methanol in that order. Each resultingsolution was then rotovapped to dryness. The hexane solids anddichloromethane solids were pooled and assayed. After the material driedin the vacuum oven overnight, 526 mg of solids were left containing 358mg of 13-desmethoxy-13-methyl-FK-520. The 13-desmethoxy-13-methyl-FK-520was found to be 68% pure by weight. Quantitation throughout all thepurification was done by UV @ 210 nm and based on a 116 mg/L13-desmethoxy-13-methyl-FK-520. Overall recovery was 86%.

EXAMPLE 7 HPLC Purification of 13-desmethoxy-13-methyl-FK520

[0195] A 25-mg sample of partially purified13-desmethoxy-13-methyl-FK520 (Example 6) was dissolved in 250 uL ofacetonitrile and insolubles were removed by centrifugation. Thesupernatant was purified by preparative HPLC using the followingconditions: column=10×250 mm InertSil ODS-3 (MetaChem), flow rate=5.0mL/min, solvent=90:10 CH₃CN/H₂O+0.1% acetic acid. Injections of 10-40 uLwere made, and the separation was monitored by UV absorption at 240 nm.The major peak for 13-desmethoxy-13-methyl-FK520 eluted at 8.7 min; Theproduct-containing fractions were pooled and evaporated to dryness undervacuum. The residue was evaporated twice from acetonitrile to removetraces of acetic acid; then lyophilized from frozen benzene and driedovernight over KOH pellets under vacuum to yield 15 mg of pure13-desmethoxy-13-methyl-FK520.

EXAMPLE 8 General Purification Protocol

[0196] The following is a general purification protocol to recover acompound of the present invention that is produced via fermentation ofhost cells. The methanolic extract from a 20-L fermentation isconcentrated to a volume of 200 mL, then poured slowly into 1500 mL ofvigorously stirred ether. The resulting suspension is stored at 4° C.overnight, then filtered. The filtrate is concentrated, and the residueis redissolved in 200 mL of ether, dried over MgSO₄, filtered, andevaporated to yield an orange-colored syrup. This is dissolved in aminimal volume of CH₂Cl₂ and loaded onto a 35-g column of SiO₂ (ISCO)equilibrated in 80:20 hexanes/acetone. The column is eluted with 80:20hexanes/acetone at a flow rate of 20 mL/min, collecting fractions ofapproximately 15 mL volume. After 10 minutes, the eluent is changed to70:30 hexanes/acetone over 5 minutes, and elution is continued anadditional 30 minutes. The fractions are analyzed by thin-layerchromatography (70:30 hexanes/acetone; staining using cerium-molybdatestain), and those fractions containing material with R_(f) valuessimilar to FK-520 are further analyzed by LC/MS. The product-containingfractions are pooled and evaporated. This material is dissolved in 1 mLof acetonitrile, diluted with 1 mL of water, and subjected topreparative HPLC using a 5 micron MetaChem InertSil ODS column (20×50mm) equilibrated in 50:50 water/acetonitrile at a flow rate of 8 mL/min.Injections of 5 mg are made, and a linear gradient from 50:50water/acetonitrile to 100% acetonitrile over 15 minutes is started after1 minute. Elution is monitored by UV absorbance at 290 nm. The analogstypically elute as two peaks: the first peak is the FK-520 analog(10,14-hemiacetal), while the seond peak is the oxepane analog(9,14-hemiacetal). The fractions containing the analog are pooled andevaporated to dryness.

[0197] Table 7 shows ¹³C-NMR data for selected compounds of theinvention. The NMR data for FK-520 is included for comparison. 13-H,15-H is 13,15-desmethoxy-FK520. 13-Me, 15-OMe is13-desmethoxy-13-methyl-FK-520. 13-H, 15-OMe is 13-desmethoxy-FK-520.13-H, 15-Me is 13-desmethoxy-15-desmethoxy-15-methyl-FK-520. 13-H, 15-Etis 13-desmethoxy-13-methyl-15-desmethoxy-15-ethyl-FK-520. TABLE 7 13H,13-H, FK-520 15-H 13-Me, 15-OMe 13-H, 15-Me 15-Me 13-H, 15-Et 13C 13C13C 13C 13C 13C 13C 13C 13C 13C position (maj) (min) (maj) (maj) (min)(maj) (min) (maj) (maj) (min)  1 169.0 168.7 169.5 169.2 168.9 169.3169.3 — 169.4 169.0  2 56.6 52.7 52.4 56.6 52.5 52.2 56.3 52.4 52.3 56.2 3 27.6 26.2 26.4 27.6 26.3 26.8 27.7 26.7 27.0 28.1  4 21.1 20.8 21.521.1 20.8 21.3 20.8 21.3 21.3 20.9  5 24.2 24.5 25.0 24.2 24.2 24.9 24.425.0 25.0 24.3  6 39.2 43.9 45.0 39.2 43.9 44.7 39.2 44.8 44.6 39.3  8164.7 165.8 165.8 164.7 166.0 165.9 164.8 — 166.0 165.4  9 196.1 192.7196.9 196.1 193.6 196.3 195.8 — 196.7 — 10 97.0 98.7 98.7 97.0 99.1 98.998.4 — 98.8 98.0 11 34.6 33.6 35.1 34.9 34.7 34.9 — 35.1 34.9 34.7 11-Me16.2 16.0 16.0 16.3 16.0 15.8 16.4 15.9 15.8 16.5 12 32.7 32.5 27.0 36.9— 34.5 — 36.4 28.8 28.9 13 73.7 73.7 30.1 30.6 30.5 27.0 — 26.8 26.927.8 13-R 56.3 56.0 na 17.2 17.1 na na na na na 14 72.9 72.3 71.1 75.575.0 73.8 70.5 75.5 73.5 71.6 15 75.2 76.6 33.0 76.7 77.7 82.1 81.6 42.241.8 15-R 57.0 57.5 na 57.0 57.1 57.9 57.7 16.3 21.4 21.3 15-R' na na nana na na na na 10.7 10.7 16 33.0 35.5 30.4 32.9 35.0 34.4 34.9 32.7 — 1726.3 26.0 32.3 26.5 26.2 27.4 27.6 29.0 28.0 — 17-Me 20.4 19.5 21.9 20.319.3 21.8 21.3 22.3 22.1 21.4 18 48.7 48.5 47.3 48.8 48.2 47.0 48.8 46.147.1 49.1 19 138.8 139.6 139.3 138.5 139.6 139.3 138.4 — 139.5 140.019-Me 15.8 15.7 17.5 15.9 16.1 17.6 16.1 17.9 17.6 16.1 20 123.1 123.3124.5 123.3 123.1 124.5 123.0 124.9 124.2 122.7 21 54.7 55.0 54.4 54.454.9 54.9 54.1 55.3 55.0 55.5 22 213.4 213.4 210.9 213.4 213.4 212.0213.3 — 212.4 — 23 43.2 43.6 46.7 43.5 43.9 44.9 44.2 45.1 44.8 43.0 2470.0 69.0 66.0 70.2 69.2 67.7 69.9 67.6 67.8 69.6 25 39.8 40.4 39.2 39.940.1 39.4 39.9 39.4 39.3 39.0 25-Me 9.6 9.9 10.0 9.6 9.9 9.9 9.4 10.010.0 8.3 26 77.2 77.9 83.2 77.2 77.9 81.0 77.2 82.1 81.8 78.1 27 132.3131.8 130.5 132.4 131.8 130.5 131.8 — 130.3 131.9 27-Me 14.1 14.2 12.314.1 14.2 12.9 14.2 12.8 12.7 14.3 28 129.7 129.6 134.8 129.8 129.6133.0 129.5 133.9 133.4 129.2 29 34.9 34.9 35.0 34.9 — 34.9 — 35.1 35.035.0 30 34.9 34.8 34.4 34.9 — 34.5 — 34.6 34.4 34.4 31 84.2 84.2 84.284.2 84.2 84.1 — 84.3 84.1 84.1 31-methoxy 56.6 56.6 56.5 56.6 56.6 56.556.6 56.8 56.5 56.6 32 73.5 73.5 73.5 73.6 73.6 73.4 73.5 73.8 73.5 73.533 31.2 31.2 31.2 31.3 31.3 31.2 — 31.4 31.2 31.2 34 30.6 30.6 30.4 30.630.6 30.4 30.6 30.8 30.4 30.6 35 24.5 24.5 23.3 24.6 24.6 23.6 24.6 24.223.2 24.6 36 11.6 11.7 11.7 11.7 11.7 11.7 11.6 11.9 11.7 11.7

EXAMPLE 9 Synthesis of 13-desmethoxy-13-methyl-18-hydroxyl-FK-520

[0198] To a mixture of 13-desmethoxy-13-methyl-FK-520 (50 mg, 0.064mmol) in 316 μL of CH₂Cl₂ was added a solution of SeO₂ (11 mg, 0.10mmol) and t-BuOOH (84 μL, 0.46 mmol) in 63 μL of CH₂Cl₂ and 6 μL of H₂Owhich were premixed until a clear solution was obtained. The reactionsolution was stirred at ambient temperature for 2 days. The solvent wasremoved and the remaining residue was purified by column chromatography(30:70 Ace-Hex) to yield 25 mg of the product as a white solid and 20 mgof the starting material.

EXAMPLE 10 Synthesis of 18-ene-20-oxa-13-desmethoxy-13-methyl-FK-520:

[0199] 24,32-bis(t-butyldimethylsilyl)-13-desmethoxy-13-methyl-FK-520:

[0200] 13-Desmethoxy-13-methyl-FK-520 (110 mg, 0.142 mmol) was suspendedin 1.77 mL of CH₂Cl₂ under a N₂ atmosphere. 2,6-Lutidine (83 mL, 0.709mmol) and TBSOTf(131 mL, 0.568 mmol) were added and the resultingsolution was stirred for 15 minutes. At this point, thin layerchromatography (“TLC”) showed a complete consumption of the startingmaterial. The reaction mixture was worked up by adding NaHCO₃ (50 mL)and extracting the product with CH₂Cl₂ (3×, 40 mL). The product waspurified by column chromatography (75:25 hexanes/ethyl acetate) to give125 mg (87%) of the desired product as a white foam. 1H NMR showed thepresence of the TBS groups.

[0201]24,32-bis(t-butyldimethylsilyl)-18-hydroxy-13-desmethoxy-13-methyl-FK-520:

[0202] A mixture of24,32-bis(t-butyldimethylsilyl)-13-desmethoxy-13-methyl-FK-520 (125 mg,0.124 mmol) in 191 mL of CH₂Cl₂ and 41 mL of EtOH was stirred with SeO₂(14 mg, 0.124 mmol) and t-BuOOH (181 mL, 0.992 mmol) at roomtemperature. After 1.5 days, another portion of SeO₂ (14 mg, 0.124 mmol)and t-BuOOH (100 mL, 0.550 mmol) were added to the reaction mixture. Thesolution was stirred for 2 more days and worked up. The solvent wasremoved and the crude reaction mixture was purified by columnchromatography (90:10 hexanes/ethyl acetate-75:25 hexanes/ethyl acetate)to give 44 mg (35%) of the product as a white foam. Mass spectroscopyshows: 1003, 871, 853.

[0203] 18-ene-20-oxa-13-desmethoxy-13-methyl-FK-520:

[0204] A mixture of24,32-bis(t-butyldimethylsilyl)-18-hydroxy-13-desmethoxy-13-methyl-FK-520(37 mg, 0.036 mmol) in 1.5 mL of acetonitrile was treated with 0.5 mL of2% aqueous HF/acetonitrile for 2.5 hours or until no starting materialwas detected by TLC. The reaction mixture was worked up by addition ofethyl acetate and aqueous saturated NaHCO₃. The product was extractedwith ethyl acetate (4×40 mL) and purified by column chromatography (1:1ethyl acetate/hexanes-60:40 ethyl acetate/hexanes) to give the titlecompound. The structure was determined by NMR spectroscopy.

EXAMPLE 11 Tris[1-(2-t-Butyldimethylsilyloxyethyl)indol-5-yl]bismuthane

[0205] 1-(2-Hydroxyethyl)-5-bromoindole:

[0206] Under a N₂ atmosphere, 2-bromoethanol (17.6 g, 141 mmol) and2-methoxypropene (10.1 g, 141 mmol) were stirred in 71 mL of THF at 0°C. for 30 minutes The resulting solution was added to a stirring mixtureof 5-bromoindole (22.83 g, 116 mmol) and 60% NaH (4.62 g, 193 mmol) in40 mL of DMF and 60 mL of THF. The solution was stirred at ambienttemperature for 4 hours. The reaction mixture was worked up by quenchingthe excess of NaH with water and removing the aqueous layer. The organiclayer was vigorously stirred with 200 mL of 2% aqueous phosphoric acidfor 5 hours when the layers were separated. The organic layer was washedwith water (2×200 mL) and the solvent removed. The residue was purifiedby column chromatography (30:70 EtOAc-Hex) to give 17 g of1-(2-hydroxylethyl)-5-bromoindole.

[0207] Tris[1-(2-t-Butyldimethylsilyloxyethyl)indol-5-yl]bismuthane:

[0208] 1-(2-hydroxylethyl)-5-bromoindole (2.08 g, 8.68 mmol),t-butyldimethylsilylchloride (1.44 g, 9.55 mmol), dimethylaminopyridine(11 mg, 0.087 mmol) and triethylamine (1.34 mL, 9.63 mmol) weredissolved in 20.7 mL of THF under N₂. The resulting solution was stirredat ambient temperature for 3 days. The mixture was cooled down andfiltered under N₂. To this solution was added n-BuLi (5.46 mL, 8.73mmol) and the solution stirred at −78° C. for 0.5 hours A solution ofBiCl₃ (958 mg, 3.04 mmol) in THF (5.0 mL) was added to the reactionmixture and the solution was stirred at −78° C. for another hour. Thereaction mixture was worked up by addition of 1 g of cellulose suspendedin 5 mL of THF and 0.65 mL of water. The supernatant was decanted anddried. The product was purified by column chromatography (5:95EtOAc-Hex) to give the product as a white solid. Mp: 119-121° C.

EXAMPLE 12 Synthesis of32-[1-(2-Hydroxyethyl)-indol-5-yl]-13-desmethoxy-13-methyl-FK-520

[0209]32-[1-(2-t-butyldimethylsilyloxyethyl)-indol-5-yl]-13-desmethoxy-13-methyl-FK-520:

[0210] A solution oftris[1-(2-t-Butyldimethylsilyloxyethyl)indol-5-yl]bismuthane (80 mg,0.077 mmol), benzoyl peroxide (17 mg, 0.071 mmol) and 2-butanone (0.965mL) were stirred at ambient temperature for 1 day. To the solution wasadded 13-desmethoxy-13-methyl-FK-520 (38 mg., 0.049 mmol) and Cu(OAc)₂(1 mg, 0.008 mmol) and the mixture was stirred for a day.- The residuewas dried and purified by column chromatography (10:90 EtOAc-Hex) togive 43 mg of product as a white foam.

[0211] 32-[1-(2-Hydroxyethyl)-indol-5-yl]-13-desmethoxy-13-methyl-FK-520(also referred to as13-desmethoxy-13-methyl-32-hydroxyethylindolyl-FK-520):

[0212] A solution of32-[1-(2-t-butyldimethylsilyloxyethyl)-indol-5-yl]-13-desmethoxy-13-methyl-FK-520(42 mg, 0.040 mmol) in 1.0 mL of MeOH and 40 μL of 1 N HCl was stirredat room temperature for about 3 hours. The reaction was worked up byremoval of the solvent followed by column chromatography (25:75EtOAc-Hex) to give 23 mg of product as a white solid.

EXAMPLE 13 Synthesis of32-[1-(2-Hydroxyethyl)-indol-5-yl]-13-desmethoxy-FK-520

[0213]32-[1-(2-t-butyldimethylsilyloxyethyl)-indol-5-yl]-13-desmethoxy-FK-520:

[0214] A solution oftris[1-(2-t-Butyldimethylsilyloxyethyl)indol-5-yl]bismuthane (81 mg,0.079 mmol), benzoyl peroxide (18 mg, 0.07 mmol) and 2-butanone (0.875mL) were stirred at ambient temperature for 1 day. To the solution wasadded 13-desmethoxy-FK-520 (40 mg., 0.051 mmol) and Cu(OAc)₂ (1.6 mg,0.0088 mmol) and the mixture was stirred for a day. The residue wasdried and purified by column chromatography (10:90 EtOAc-Hex) to give 23mg of product as a white foam.

[0215] 32-[1-(2-Hydroxyethyl)-indol-5-yl]-13-desmethoxy-FK-520:

[0216] A solution of32-[1-(2-t-butyldimethylsilyloxyethyl)-indol-5-yl]-13-desmethoxy-FK-520(23 mg, 0.022 mmol) in 0.89 mL mL of MeOH and 22 μL of 1 N HCl wasstirred at room temperature for about 3 hours. The reaction mixture wasdiluted with NaHCO₃ and extracted with CH₂CL₂ (3×, 50 mL). The organiclayer was removed and the residue was purified by column chromotagraphy(25:75 EtOAc-Hex) to give the product as a white solid.

EXAMPLE 14 FKBP-12 Binding Assay

[0217] Calcineurin (“>95% pure by SDS/PAGE”) was obtained fromCalbiochem. Neutravidin® coated strip plates, pre-blocked withSuperBlock® were obtained from Pierce. [³H]FK506 (87 Ci/mmol), labelledby saturation of the allyl group, was obtained from New England Nuclear.Synthetic RII peptide (⁺H₃N-D-L-D-V—P—I—P-G-R—F-D-R—R—V—S—V-A-A-E-CO₂ ⁻)was obtained from Peptides International (Louisville, Ky.;www.pepnet.com). [γ-³²P]ATP (6000 Ci/mmol) was obtained from Pharmacia.ATP, cAMP, the catalytic subunit of protein kinase A, bovine braincalmodulin and human FKBP, expressed in E. coli were obtained fromSigma.

[0218] Wells of Neutravidin® coated strip plates were coated with 100 μLof 1 μM biotinylated FKBP-12 (100 pmol) in 20 mM sodium phosphate, pH7.4 for 2-4 hours at room temperature. Wells were rinsed with 3×200 μLPBS containing 0.2% Tween 20 (PBS-tween), then filled with 100 uL ofPBS-tween containing 0.5 μM [³H]FK-506 (4-5000 dpm/pmol, 150-200,000dpm/assay) and 0-10 μM of competing ligand (unlabelled FK-506, FK-520,or 13-methyl-13-desmethoxy-FK-520). The mixture was incubated for 2hours at 0° C., the solution was aspirated from the wells, and each wellwas quickly washed with 300 uL of ice cold PBS-tween. Wells were brokenapart, placed in scintillation vials with 10 mL of Scintiverse BD andbound radioactivity was quantitated by scintillation counting. Data werefit to a competitive binding equation in which the K_(d) of [³H]FK-506was assumed to be 0.4 nM:${dpmbound} = {\max \quad {dpmbound}\quad \left( {1 - \left( \frac{\left\lbrack {{competing}\quad {ligand}} \right\rbrack}{\left( {\left\lbrack {{competing}\quad {ligand}} \right\rbrack + {K_{d}\left( {1 + {{\left\lbrack {\left\lbrack {}^{3}H \right\rbrack {FK506}} \right\rbrack/0.4}\quad {nM}}} \right.}} \right.} \right)} \right)}$

[0219] to calculate the K_(d).

EXAMPLE 15 Calcineurin Binding Assay

[0220] A peptide corresponding to residues 81-99 of the regulatorysubunit of bovine type II cAMP-dependent protein kinase (“RII peptide”)has been shown to be an optimal minimal substrate for calcineurin. Thecatalytic subunit of protein kinase A was used to transfer the labeledphosphate from [γ-³²P]ATP to serine-15 of the RII peptide. ATP wasfreshly dissolved in kinase buffer (40 mM MES, pH 6.5, 0.4 mM EGTA, 0.8mM EDTA, 4 mM MgCl₂, 0.1 mM CaCl₂, 0.1 mg/mL BSA), quantitatedspectrophotometrically (ε₂₅₉=15.4 mM⁻¹cm⁻¹) and used to dilute thespecific activity of [γ-³²P]ATP to ˜5000 dpm/pmol. Phosphorylationreactions (200 uL) were performed in kinase buffer and contained 162 uM[γ-³²P]ATP, 150 uM RII peptide, 0.2 uM cAMP, 25 ug/mL (160 units)protein kinase A catalytic subunit. Reaction mixtures were incubated at30° C. for 3.5 hours, then purified by solid phase extraction using a 3mL (200 mg) Bakerbond C18 cartridge. The cartridge was equilibrated with3 mL 30% acetonitrile/0.1% TFA followed by 5 mL of 0.1% TFA, thereaction mixture was loaded onto the column, washed with 20 mL of 0.1%TFA, and the product was eluted with 4×1 mL of 30% acetonitrile/0.1%TFA. Fractions were collected and monitored by scintillation counting.Phosphopeptide containing fractions were pooled and evaporated todryness by vacuum centrifugation. The phosphopeptide product wereanalyzed by HPLC using a 250×4.6 mm Intertsil C18 column (Metachem) at 1mL/min with a gradient from H₂0/pH 3 w/H₃PO₄ to 50% acetonitrile over 45minutes. Separation between the RII peptide and phosphopeptide wasmonitored at 225 nm. Reversed phase HPLC using 0.1% TFA in a gradient ofwater to 50% acetonitrile over 45 minutes failed to resolve the startingpeptide from the phosphopeptide product; however, the same column usinga gradient from water/pH 3.0. w/H₃PO₄ to 50% acetonitrile over 45minutes separated these compounds (RII peptide elutes at 17.4 min,phospho-RII peptide elutes at 19 minutes). Using this system, thephosphopeptide used in calcineurin assays was shown to contain <1% ofnonphosphorylated peptide and have a radiochemical purity>95%.

[0221] To determine reaction parameters for initial rate assays,reaction mixtures (75 uL) containing 0-60 nM calcineurin and 80 nMcalmodulin in calcineurin assay buffer (40 mM Tris, pH 7.5, 6 mM MgCl₂,0.1 mM CaCl₂, 0.1% BSA and 0.5 mM DTT) were initiated by addition of 1uM phosphopeptide. The reactions were incubated at 30° C., and 10-25 uLaliquots were removed at 3, 12, 23, 47, 100, and 250 minutes andquenched with 0.5 mL 100 mM KPO₄/5% trichloroacetic acid (“TCA”). ³²PO₄was isolated from unreacted phosphopeptide using a dedicated 0.5 mL (bedvolume) Dowex AG50×8 for each sample. The columns were prepared byresuspending the resin in water such that there was one volume of waterabove each volume of settled resin. The slurry (1 mL) was then pipettedinto each column, followed by 10 mL of water. The bed volume wasverified, then each column was washed with 1 mL 1 N NaOH, 2 mL 1 N HCl,and 4 mL water. Quenched reaction mixtures were applied to the columns,washed twice with 750 uL of water and the eluate was collected directlyinto scintillation vials. Scintiverse BD (15 mL) was added to each vial,and ³²PO₄ was quantitated by scintillation counting.

[0222] The K_(i) values for calcineurin phosphatase activity weredetermined in reaction mixtures containing 40 mM Tris, pH 7.5, 6 mMMgCl₂, 0.1 mM CaCl₂, 0.1% BSA and 0.5 mM DTT, 15 nM calcineurin and 30nM calmodulin. For inhibition by FK-506 and FK-520, these molecules wereincluded at ˜6 uM and FKBP-12 concentration was varied from 0.01 uM to2.5 uM. Following a 30 minute preincubation, reactions were initiated byaddition of the phospho-RII peptide substrate to 1 uM and incubated at30° C. Aliquots were removed at 3 and 35 minutes, quenched, and PO₄release was measured as described above. Binding data were fit to anequation which corrects for depletion of the FKBP•compound complex bycalcineurin binding:$V_{i} = {V_{o}\left( {1 - \left( \frac{\left( {{\lbrack E\rbrack t} + {\lbrack S\rbrack t} + {Ki}} \right) - \sqrt{{\left( {{\lbrack E\rbrack t} + {\lbrack S\rbrack t} + {Ki}} \right)2} - {{4\lbrack E\rbrack}{t\lbrack S\rbrack}t}}}{{2\lbrack E\rbrack}t} \right)} \right.}$

[0223] Where

[0224] V_(i) is the observed rate, [S]_(t) is the total concentration ofFKBP•compound complex, [E]_(t) is the total amount of calcineurin used,K_(i) is the inhibition constant, and V_(o) is the rate in the absenceof inhibition.

[0225] The rate of phosphate hydrolysis in an illustrative set ofcalcineurin phosphatase assays was linear with calcineurin concentrationin the range examined (0-60 nM). When 15 nM calcineurin was used, thereaction was linear for 45 min, using ˜10% of the substrate.

EXAMPLE 16 Metabolism of13-desmethoxy-13-methyl-32-(2-hydroxyethylindolyl)-FK-520

[0226] A mixture containing FK species and a NADPH regenerating systemwas pre-incubated at 37° C. for 10 minutes before the reaction wascommenced by the addition of the P-450 supersomes. The finalconcentrations of the components were: 100 mM potassium phosphate, pH7.4; 3.3 mM MgCl₂; 3.3 mM glucose-6-phosphate; 1.3 mM NADP; 0.4 U/mLglucose-6-phosphate dehydrogenase; 200 pmole P-450/mL; and 20 μM FKspecies. Control reactions contained “mock” supersomes without 3A4 P-450activity. Human 3A4 P-450+Reductase supersomes (cat # P207) and vectorcontrol “mock” supersomes without 3A4P-450 (cat # P201) were obtainedfrom Gentest Corporation (Woburn, Mass.).

[0227] Following addition of supersomes or mock supersomes, reactionswere terminated at 0 minutes (immediately upon addition of P-450) and 30minutes by addition of acetonitrile containing 0.1% acetic acid to 20%final acetonitrile concentration, followed by freezing immediately ondry ice. For reactions containing the13-desmethoxy-13-methyl-32-(2-hydroxyethylindolyl)-FK analog, an equalvolume of MeOH was added following addition of acetonitrile and thesamples were immediately frozen on dry ice. The samples were clarifiedby centrifugation at 13,000 rpm for 5 minutes at 4° C. in a microcentrifuge prior to HPLC analysis.

[0228] The following HPLC program was employed: Column=MetaChem 0.46×15cm intersil C18 column (5 μm); Solvent A=0.1% HOAc in water; SolventB=0.1% HOAc in acetonitrile. Detection=UV (210 nm) and ELSD. Gradient:Equilibration with 20% B; injection (up to 1 ml), hold at 20% B for 5minutes; linear gradient to 50% B in 5 minutes; linear gradient to 100%B in 20 minutes; to 20% B in 1 minute; hold at 20% B for 10 minutes toequilibrate.

[0229] There was no change in peak area after 30 minutes when the FKanalogs were incubated with mock supersomes. Hence, there was no timedependent loss of the parent compound due to (a) adsorption to proteinsand membranes in the supersome preparation or (b) due to non-3A4 P-450activities in the supersome preparation.

EXAMPLE 17 Biological Activities of Immunosuppressive Agents of thePresent Invention

[0230] Table 8 summarizes the results of a FKBP binding assay,calcineurin inhibition assay and a P450 stability assay for FK-520 and aselect number of the compounds of the present invention. Protocols forthese assays are described in Examples 14-16. TABLE 8 P450% FKBPCalcineurin Stability Binding Inhibition (at Compound (Kd) Ki 30 min)FK-520  0.4 nM    49 nM 46 13-desmethoxy-FK-520  0.4 nM    32 nM 3513-desmethoxy-13-methyl-FK-520  1.6 nM   940 nM 2632-[1-(2-hydroxyethyl)-indol-5-yl]- 16.3 nM  <15 nM 7213-desmethoxy-FK-520 32-[1-(2-hydroxyethyl)-indol-5-yl]- 10.4 nM    22nM 77 13-desmethoxy-FK-520

EXAMPLE 18 Biological Activities of Neurotrophic Agents of the PresentInvention

[0231] Table 9 summarizes the results of nerve growth as using SH-SY5Yhuman neuroblastoma cells according to Gold et al., Exp Neuro, 147(2):269-87 (1997) which is incorporated herein by reference. The assaymeasures the mean neurite length in μM that were induced at 0.1 nM and10 nM of the compound of interested after 96 or 168 hours. TABLE 9Neurite Length Neurite Length Neurite Length Neurite Length at 0.1 nM 96h at 0.1 nM 168 h at 10 nM 96 h at 10 nM 168 h Compound (μM) (μM) (μM)(μM) No treatment  80  95 — — Nerve Growth Factor 143 169 — —18-hydroxy-13- 168 191 132 165 desmethoxy-13-methyl- FK-52018-ene-20-oxa-13- 144 184 104 135 desmethoxy-13-methyl- FK-520

[0232] 18-Hydroxy-13-desmethoxy-13-methyl-FK-520 is an ideal candidatefor a neurotrophic agent because it does not also possessimmunosuppressive activity. Although this compound binds FKBP with aK_(d) of approximately 1.0 nM, it has a K_(i) in the calcineurininhibition assay of greater than about 14,000 nM.

[0233] All scientific and patent publications referenced herein arehereby incorporated by reference. The invention having now beendescribed by way of written description and example, those of skill inthe art will recognize that the invention can be practiced in a varietyof embodiments, that the foregoing description and example is forpurposes of illustration and not limitation of the following claims.

What is claimed is:
 1. A lactone having a K_(d) in a FKBP binding assayof approximately equal to or less than 1 μM and including a fragment

as part of its structure wherein R⁴ and R⁵ are each independentlyselected from the group consisting of hydrogen, hydroxyl, methyl, ethyl,and methoxy, provided that at least one of R⁴ and R⁵ is hydrogen, methylor ethyl.
 2. The lactone as in claim 1 of the formula

wherein: R is hydroxyl; R¹ is selected from the group consisting ofhydrogen, methyl, propyl, ethyl and allyl; R² and R³ are eachindependently hydrogen or hydroxyl; R⁴ and R⁵ are each independentlyselected from the group consisting of hydrogen, hydroxyl, methyl, ethyl,and methoxy, provided that at least one of R⁴ and R⁵ is hydrogen,methyl, or ethyl; R⁶ is selected from a group consisting

wherein R⁷ is selected from the group consisting of hydrogen, hydroxyl,methyl, ethyl, and methoxy and R⁸ is selected from a group consisting ofhydrogen, hydroxyl, C₁-C₁₀ alkyl, C₁-C₁₀ alkoxy, aryl, aryloxy,arylalkyl, and arylalkoxy; and, a double bond exists between carbon-19and carbon-20, or a double bond exists between carbon-18 and carbon-19and R and R² together are oxygen forming a lactone ring.
 3. The lactoneas in claim 1 of the formula

wherein: R is hydroxyl; R¹ is selected from the group consisting ofhydrogen, methyl, ethyl, and allyl; R² and R³ are each independentlyhydrogen or hydroxyl; R⁴ and R⁵ are each independently selected from thegroup consisting of hydrogen, hydroxyl, methyl, ethyl, and methoxy,provided that at least one of R⁴ and R⁵ is hydrogen, methyl, or ethyl;R⁷ and R⁸ are each independently selected from a group consisting ofhydrogen, hydroxyl, C₁-C₁₀ alkyl, C₁-C₁₀ alkoxy, aryl, aryloxy,arylalkyl, and arylalkoxy; and, a double bond exists between carbon-19and carbon-20, or a double bond exists between carbon-18 and carbon-19and R and R² together are oxygen forming a lactone ring.
 4. The lactoneas in claim 3 wherein R is hydroxyl; R¹ is selected from the groupconsisting of hydrogen, methyl, ethyl, and allyl; R² and R³ are eachindependently hydrogen or hydroxyl; R⁴ and R⁵ are each independentlyselected from the group consisting of hydrogen, hydroxyl, methyl, ethyl,and methoxy, provided that at least one of R⁴ and R⁵ is hydrogen,methyl, or ethyl; R⁷ is selected from the group consisting of hydrogen,hydroxyl, methyl, ethyl, C₁-C₅ alkoxy and aryloxy; R⁸ is selected from agroup consisting of hydrogen, hydroxyl,

wherein R⁹ is selected from the group consisting of hydrogen, hydroxyl,halide, C₁-C₅ alkyl, C₁-C₅ hydroxyalkyl, and C₁-C₅ alkoxy; andoptionally, a double bond exists between carbon-19 and carbon-20, or adouble bond exists between carbon-18 and carbon-19 and R and R² togetherare oxygen forming a lactone ring.
 5. A compound of the formula

wherein R¹ is ethyl or allyl; R³ is hydrogen or hydroxyl; R⁴, R⁵ and R⁷are each independently selected from the group consisting of hydrogen,hydroxy, methyl, ethyl, and methoxy; and R⁸ is selected from a groupconsisting of hydrogen, hydroxyl, C₁-C₅ alkoxy, and heteroaryloxy,provided that at least one of R⁴ and R⁵ is hydrogen, methyl or ethyl. 6.The compound as in claim 5 wherein R¹ is ethyl or allyl; R³ is hydrogenor hydroxyl; R⁴ and R⁵ are each independently selected from the groupconsisting of hydrogen, hydroxyl, methyl, ethyl, and methoxy; R⁷ ismethoxy; and, R⁸ is hydroxyl or

where R⁹ is selected from the group consisting of hydrogen, hydroxyl,hydroxymethyl and hydroxyethyl.
 7. The compound as in claim 6 of theformula

wherein R¹ is ethyl or allyl; and R⁴ and R⁵ are each independentlyselected from the group consisting of hydrogen, hydroxyl, methyl, ethyl,and methoxy.
 8. The compound as in claim 7 wherein R¹ is ethyl; R⁴ ismethoxy; and, R⁵ is selected from the group consisting of hydrogen,hydroxyl, methyl, and ethyl.
 9. The compound as in claim 7 wherein R¹ isethyl; R⁴ is ethyl; and, R⁵ is selected from the group consisting ofhydrogen, hydroxyl, methyl, and ethyl.
 10. The compound as in claim 6 ofthe formula

wherein R¹ is ethyl or allyl; and R⁴ and R⁵ are each independentlyselected from the group consisting of hydrogen, hydroxyl, methyl, ethyl,and methoxy.
 11. The compound as in claim 6 of the formula

wherein R¹ is ethyl or allyl; and R⁴ and R⁵ are each independentlyselected from the group consisting of hydrogen, hydroxyl, methyl, ethyl,and methoxy.
 12. The compound as in claim 11 wherein R¹ is ethyl; R⁴ ismethoxy; and, R⁵ is selected from the group consisting of hydrogen,hydroxyl, methyl, and ethyl.
 13. The compound as in claim 11 wherein R¹is ethyl; R⁴ is ethyl; and, R⁵ is selected from the group consisting ofhydrogen, hydroxyl, methyl, and ethyl.
 14. The compound as in claim 6 ofthe formula


15. The compound as in claim 6 of the formula


16. The compound as in claim 6 of the formula


17. The compound as in claim 6 of the formula


18. The compound as in claim 6 of the formula


19. The compound as in claim 6 of the formula


20. The compound as in claim 6 of the formula


21. The compound as in claim 6 of the formula


22. The compound as in claim 6 of the formula


23. The compound as in claim 6 of the formula


24. The compound as in claim 6 of the formula


25. The compound as in claim 6 of the formula


26. The compound as in claim 6 of the formula


27. The compound as in claim 6 of the formula


28. A recombinant host cell selected from the group consisting ofKOS-45-170 (PTA-1811); KOS-60-135 (PTA-1810); KOS132-188; KOS132-191;KOS156-25; KOS156-9A; KOS156-9B; KOS156-26; KOS156-33A; KOS156-33B; andKOS156-33C.
 29. A compound produced by a recombinant host cell selectedfrom the group consisting of KOS-45-170 (PTA-1811); KOS-60-135(PTA-1810); KOS132-188; KOS132-191; KOS156-25; KOS156-9A; KOS156-9B;KOS156-26; KOS156-33A; KOS156-33B; and KOS 156-33C.
 30. The compound ofclaim 29 of the formula

produced by KOS45-170.
 31. The compound of claim 29 of the formula

produced by KOS60-135.